Imipridones for gliomas

ABSTRACT

Imipridones selectively modulate Class A G protein-coupled receptors (GPCRs), such as the D2-like subfamily of dopamine receptors, and are useful for treating conditions and disorders in need of such modulation, such as cancers. Specifically, the cancer is a midline glioma, a cancer having a histone H3 mutation, or both. In addition, methods of identifying whether a subject having these conditions, is likely to be responsive to a treatment regimen, such as imipridone administration, are provided. Furthermore, methods of assessing the effectiveness of a treatment regimen, such as imipridone administration, monitoring, or providing a prognosis for a subject with these condition are also provided.

BACKGROUND OF THE INVENTION

ONC201 (7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido [3,4-e]pyrimidin-5(1H)-one) is the founding member of aclass of anti-cancer compounds called imipridones that is in Phase IIclinical trials in multiple advanced cancers. Since the discovery ofONC201 as a p53-independent inducer of TRAIL gene transcription,preclinical studies have determined that ONC201 has anti-proliferativeand pro-apoptotic effects against a broad range of tumor cells but notnormal cells. The mechanism of action of ONC201 involves engagement ofPERK-independent activation of the integrated stress response, leadingto tumor upregulation of DR5 and dual Akt/ERK inactivation, andconsequent Foxo3a activation leading to upregulation of the death ligandTRAIL. ONC201 is orally active with infrequent dosing in animal models,causes sustained pharmacodynamic effects, and is not genotoxic. Thefirst-in-human ONC201 clinical trial in advanced aggressive refractorysolid tumors confirmed that it is well-tolerated.

The discovery of H3 K27M as an oncogenic mutation occurred in thecontext of midline gliomas that involve the thalamus, pons, or spinalcord. H3 K27M refers to a specific mutation in histone H3 proteins. Dueto the location of these tumors, areas of the brain involved in criticalphysiological functions, these tumors have historically been inoperable(especially in the brain stem where the pons is located). This meansthat until recently, midline gliomas such as diffuse intrinsic pontineglioma (DIPG) were diagnosed solely on a radiographic basis. Advances inneurosurgical techniques and increased parental consent to post-mortemtumor tissue retrieval led to the availability of sufficientbiospecimens that enabled systematic genomic evaluations of DIPG andother midline gliomas. Gliomas in the midline of the brain belong to themost aggressive types of primary malignant brain cancers. The diseasearises from glial cells, which are cells that form the tissue thatsurrounds and protects other nerve cells found within the brain andspinal cord.

Standard therapy for midline gliomas involves neurosurgery, whenfeasible, followed by fractionated external beam radiotherapy. Due tolocation in the brain, aggressiveness and low survival time, gliomas inthe midline of the brain are considered as part of the most lethal formsof cancer.

No medical therapies have been shown to prolong survival in H3 K27Mmutant adult and pediatric glioma patients. Standard-of-care treatmentfor DIPG, 55Gy focal radiation fractionated over 6 weeks, is associatedwith a 9-11-month overall survival. For adult H3 K27M glioma, thedisease is often treated with the same regimen as glioblastoma thatinvolves radiation with concomitant and maintenance temozolomide.Despite its use to treat this newly defined disease in adults, theefficacy of this regimen has not been evaluated specifically in adultmutant H3 K27M glioma patients.

The functions of histones are predominantly protein-DNA andprotein-protein interactions and they do not function as enzymes, whichhave represented the bulk of targeted cancer therapy (kinases, HDACinhibitors, etc.). Thus, no therapies directly target the mutant H3proteins itself (such as the case for mutant BRAF); instead, therapeuticefforts have focused on targeting features of tumor cells with H3 K27Mmutations, such as their epigenetic- and transcription-dependency.Inhibition of proteins involved in epigenetics such as histonede-acetylates, histone de-methylases, or bromodomains have yieldedefficacy in preclinical models, however their ability to improveclinical outcome has not been shown.

A major challenge for effective H3 K27M glioma treatments is thattherapeutics must penetrate the blood-brain barrier, a rare feature ofcurrent cancer therapies. This requirement is further enhanced by thelocation of these tumors in midline brain structures, which have beenshown to be more difficult to penetrate than other brain locations. TheH3 K27M mutation also tends to occur in midline gliomas where dopamineis present and DRD2 expression is prevalent in the tumor environment.

The lack of treatments for H3 K27M glioma leave a large unmet medicalneed with respect to disease control, symptom relief, and survivalrates. Patients with recurrent disease following radiation, are leftwith no treatment options with a demonstrated survival benefit.

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided herein are compounds of formula (10):

wherein R₁ and R₂ are independently selected from H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl,aralkoxy, aralkylthio, and acyl radicals. In one embodiment, when R₁ isCH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂is CH₂-(2-CH₃-Ph) (ONC201). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph) (ONC206). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph) (ONC212). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(3,4-di F-Ph) (ONC213). In one embodiment, R₁ is CH₂-(3,4-di-Cl-Ph)and R₂ is CH₂-(4-CF₃-Ph) (ONC234). In one embodiment, R₁ isCH₂-3-thienyl and R₂ is CH₂-(4-CF₃-Ph) (ONC236).

In another aspect, provided herein are methods of treating or preventingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount compound (1)

or a pharmaceutically acceptable salt thereof, wherein the cancer is amidline glioma having a histone H3 K27M mutation.

In another aspect, provided herein are methods of treating or preventingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof,wherein the cancer has a histone H3 mutation.

In another aspect, provided herein are methods of treating or preventingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof,wherein the cancer is a midline glioma.

BRIEF DESCRIPTION OF THE DRAWINGS

The above summary, as well as the following detailed description ofembodiments of the invention, will be better understood when read inconjunction with the appended drawings. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities shown. In the drawings:

FIG. 1. Antagonism of dopamine receptors (DRD1, DRD2S, DRD2L, DRD3,DRD4, and DRD5) by ONC201.

FIG. 2. Tumor type sensitivity of the Genomic of Drug Sensitivity inCancer program (GDSC) cell line collection. The average sensitivity wasdetermined by average estimated IC₅₀ values from cell viability assaysconducted at 72 hours post-treatment. Numbers above the bar indicatesindicate the number of cell lines per tumor type.

FIG. 3. GBM cell lines with higher DRD2 or lower DRD5 expression aremore responsive to ONC201. (A) Inhibition of NCI60 GBM cell lines as afunction of ONC201 concentration. (B) Log ONC201 GI₅₀ (M) vs DRD2expression for each GBM cell line. R²=0.8707. (C) Low DRD5 expressionsignificantly correlates with improved ONC201 efficacy in NCI60 panel ofcancer cell lines.

FIG. 4. ONC206 and ONC212 demonstrated anti-cancer efficacy acrossvarious tumor types in the NCI60 cancer cell line panel. ONC203 is aninactive negative control.

FIG. 5. Bone cancer is more responsive to ONC206 than ONC201.

FIG. 6. Ewing's sarcoma is the most ONC206 responsive bone cancersubtype.

FIG. 7. ONC206 anti-cancer efficacy is in the nanomolar range in 14 outof 16 Ewing's sarcoma cell lines. ONC206 demonstrated superior efficacycompared to ONC201 in all cell lines

FIG. 8. ONC212 induced cell death in cancer cells (HCT116) but notnormal cells (MRC5) at nanomolar concentrations.

FIG. 9. ONC212 induces the integrated stress response and inhibitsAkt/ERK phosphorylation at nanomolar concentrations and at earlier timepoints compared to ONC201.

FIG. 10. ONC212 demonstrates oral and IP anti-cancer efficacy inxenograft mouse models of colorectal and breast cancer.

FIG. 11. Leukemia is more responsive to ONC212 than ONC201.

FIG. 12. ONC212 demonstrates anti-cancer efficacy (and superior efficacycompared to ONC201) in the nanomolar range in 55 leukemia cell linesregardless of subtype.

FIG. 13. A subject with recurrent glioblastoma (Example 14). (A) Tumorsize relative to baseline (%) of total tumor burden in the subject. Onecycle is 3 weeks. (B) Contrast MRI scans at baseline, 21, 27 and 36weeks post-ONC201 initiation of one of 2 malignant lesions.

FIG. 14. Anti-cancer efficacy of ONC212 in acute myeloid leukemia (AML)cell lines. (A) Comparison of cell viability of MV411 AML cells treatedwith ONC212 or cytarabine. (B) Comparison of cell viability of MOLM14,MV411 AML cells, MRC5 lung fibroblasts and Hs27a bone marrow cellstreated with ONC212. (C) Cell viability of MOLM14 and MV411 AML cellstreated with ONC212 (250 nM) for 4, 8, 24, 48, 72 and 96 h.

FIG. 15. ONC212 efficacy in ONC201-resistant AML xenograft model (MV411AML cells (5×10⁶) subcutaneously implanted in the flanks of athymic nudemice). ONC212 and ONC201 were administered orally (PO) as indicated.Tumor volume (A and B) and body weight (C) (n=10) was measured onindicated days. * represents p<0.05 relative to vehicle.

FIG. 16. ONC206 efficacy in Ewing's sarcoma xenograft model (MHH-ES-1Ewing's sarcoma cells (5×10⁶) subcutaneously implanted in the flanks ofathymic nude mice). ONC206 (PO) and methotrexate (IV) were administeredon day 1 and day 13 as indicated. Tumor volume (A) and body weight (B)(n=4) was measured on indicated days.

FIG. 17. ONC213 demonstrated in vitro anti-cancer potency inHCT116/RPMI8226 cancer cells similar to ONC212, but in vitro toxicity tonormal cells was reduced compared to ONC212.

FIG. 18. The subject with recurrent H3 K27M thalamic glioblastoma ofExample 14 has a durable objective response. (A) Relative overall tumorsize (%) in the subject. (B) Contrast MRI scans at baseline, and 17months of treatment with ONC201.

FIG. 19. Immune induction correlates with tumor shrinkage inglioblastoma.

FIG. 20. A 74-year-old woman with recurrent H3 K27M glioblastoma. Firston-treatment 8 week MRI shows complete disappearance of tumor lesions.

FIG. 21. A 10-year-old girl with H3 K27M diffuse intrinsic pontineglioma has improvement in facial palsy and shrinkage of lesion after 16doses.

FIG. 22. A 3-year-old girl with H3 K27M diffuse intrinsic pontineglioma. First on-treatment 6 week MRI shows stable tumor lesion.

FIG. 23. Progression-free survival of patients with recurrent high gradeglioma present at baseline by MRI before initiating ONC201 therapy. Thecohort is divided into two groups: one with known H3 K27M mutation (redcurve) and the other with wild-type or unknown H3 status (blue curve).

DETAILED DESCRIPTION OF THE INVENTION

Scientific and technical terms used here are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found and used in context in various standard referencesillustratively including J. Sambrook and D. W. Russell, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press;3^(rd) Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts of RNAiTechnology, DNA Press LLC, Eagleville, P A, 2003; Herdewijn, P. (Ed.),Oligonucleotide Synthesis: Methods and Applications, Methods inMolecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K.Vintersten, R. Behringer, Manipulating the Mouse Embryo: A LaboratoryManual, 3^(rd) edition, Cold Spring Harbor Laboratory Press; Dec. 15,2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells:methods and protocols in Methods Mol Biol. 2002; 185, Humana Press;Current Protocols in Stem Cell Biology, ISBN: 9780470151808, as well asU.S. Pat. No. 8,673,923. The content of each of the references above ishereby incorporated by reference in its entirety.

The term “substituted” means that any one or more hydrogens on thedesignated atom is replaced with a selection from the indicated group,provided that the designated atom's normal valency is not exceeded, andthat the substitution results in a stable compound. When a substituentis keto (i.e., ═O), then 2 hydrogens on the atom are replaced. Ketosubstituents are not present on aromatic moieties. Ring double bonds aredouble bonds that are formed between two adjacent ring atoms (e.g., C═C,C═N, or N═N).

When a variable (e.g., R⁴) occurs more than one time in a constituent orformula for a compound, its definition at each occurrence is independentof its definition at every other occurrence. Thus, for example, if agroup is shown to be substituted with 0-3 R⁴ moieties, then the groupmay optionally be substituted with up to three R⁴ moieties and R⁴ ateach occurrence is selected independently from the definition of R⁴.Also, combinations of substituents and/or variables are permissible, butonly if such combinations result in stable compounds.

When an atom or chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it will be appreciated that this is meant toencompass each number within the range, as well as all intermediateranges. For example, “C₁₋₆ alkyl” is meant to include alkyl groups with1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5,3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” includes both branched and straight-chain saturatedaliphatic hydrocarbon groups having the specified number of carbonatoms. For example, C₁₋₆ alkyl includes C₁, C₂, C₃, C₄, C₅, and C₆ alkylgroups. Non-limiting examples of alkyl include methyl, ethyl, n-propyl,i-propyl, n-butyl, isobutyl s-butyl, t-butyl, n-pentyl, s-pentyl,neopentyl and n-hexyl. In certain cases, a straight or branched chainalkyl has six or fewer carbon atoms in its backbone (e.g., C₁-C₆ for astraight chain, C₃-C₆ for a branched chain); in other cases, a straightor branched chain alkyl has four or fewer carbon atoms. Likewise,cycloalkyls may have from three to eight carbon atoms in their ringstructure; in some cases, cycloalkyls have five or six carbons in thering structure. Most preferred is C₁₋₆ alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

The term “substituted alkyl” means alkyl as defined above, substitutedby one, two or three substituents selected from halogen, —OH, alkoxy,—NH₂, —N(CH₃)₂, —C(═O)OH, trifluoromethyl, —C═N, —C(═O)O(C₁-C₄)alkyl,—C(═O)NH₂, —SO₂NH₂, —C(═NH)NH₂, and —NO₂, preferably containing one ortwo substituents selected from halogen, —OH, alkoxy, —NH₂,trifluoromethyl, —N(CH₃)₂, and —C(═O)OH, more preferably selected fromhalogen, alkoxy and —OH. Non-limiting examples of substituted alkylsinclude 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

Unless the number of carbons is otherwise specified, “lower alkyl” is analkyl group, as defined above, but having from one to six carbon atoms,preferably one to four, in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of 2-6 carbon atoms and preferably2-4 carbon atoms.

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includesstraight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched-chainalkenyl groups, cycloalkenyl (e.g., alicyclic) groups (e.g.,cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. In certain cases,a straight or branched chain alkenyl group has six or fewer carbon atomsin its backbone (e.g., C₂-C₆ for a straight chain, C₃-C₆ for a branchedchain). Likewise, cycloalkenyl groups may have from three to eightcarbon atoms in their ring structure; in some cases, cycloalkenyl groupshave five or six carbons in the ring structure. The terms “C₂-C₆” and“C₃-C₆” includes alkenyl groups containing two to six carbon atoms andthree to six carbon atoms, respectively.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight-chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), branched-chain alkynyl groups, andcycloalkyl or cycloalkenyl substituted alkynyl groups. In certain cases,a straight or branched chain alkynyl group has six or fewer carbon atomsin its backbone (e.g., C₂-C₆ for a straight chain, C₃-C₆ for a branchedchain). The terms “C₂-C₆” and “C₃-C₆” includes alkynyl groups containingtwo to six carbon atoms and three to six carbon atoms, respectively.

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromaticradical, where each of the atoms forming the ring (i.e. skeletal atoms)is a carbon atom. In some cases, the cycloalkyl group is saturated orpartially unsaturated. In other cases, the cycloalkyl group is fusedwith an aromatic ring. Cycloalkyl groups include groups with from 3 to10 ring atoms. Examples of cycloalkyl groups include, but are notlimited to, the following moieties:

Monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include,but are not limited to, tetrahydronaphthyl, indanyl, andtetrahydropentalene. Polycyclic cycloalkyls include adamantine andnorbornane. The term cycloalkyl includes “unsaturated nonaromaticcarbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both ofwhich refer to a nonaromatic carbocycle as defined herein, whichcontains at least one carbon carbon double bond or one carbon carbontriple bond.

The term “cycloalkylalkyl” refers to an alkyl group substituted by acycloalkyl group. Example cycloalkylalkyl groups includecyclopropylalkyl, cyclohexylalkyl.

The term “heterocycloalkyl” refers to a non-aromatic heterocycle whereone or more of the ring-forming atoms is a heteroatom such as an O, N,or S atom. Heterocycloalkyl groups include mono- or polycyclic (e.g.,having 2, 3 or 4 fused rings) ring systems, as well as spirocycles.Example heterocycloalkyl groups include morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, and imidazolidinyl. Also included in thedefinition of heterocycloalkyl can be moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thenonaromatic heterocyclic ring, for example, quinolyl, isoquinolyl, andbenzo derivatives of heterocycles. A heterocycloalkyl group having oneor more fused aromatic rings are attached though either the aromatic ornon-aromatic portion. Also included in the definition ofheterocycloalkyl are moieties where one or more ring-forming atoms canbe substituted by 1 or 2 oxo or sulfido groups. In some cases, theheterocycloalkyl group has from 1 to about 20 carbon atoms, and infurther case from about 3 to about 20 carbon atoms. In some cases, aheterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about7, or 5 to 6 ring-forming atoms. In some cases, a heterocycloalkyl grouphas 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some cases, aheterocycloalkyl group contains 0 to 3 double bonds. In some cases, aheterocycloalkyl group contains 0 to 2 triple bonds.

The term “heterocycloalkylalkyl” refers to an alkyl group substituted bya heterocycloalkyl. Example heterocycloalkylalkyls includemorpholinoalkyl and piperazinylalkyl.

The term “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or4 fused rings) aromatic hydrocarbons, such as phenyl, naphthyl,anthracenyl, phenanthrenyl. In some cases, an aryl group has from 6 toabout 20 carbon atoms.

The term “arylalkyl” refers to an alkyl group substituted by an arylgroup. Example arylalkyl groups include benzyl and phenylethyl.

The term “heteroaryl” refers to an aromatic heterocycle having at leastone heteroatom ring member such as an O, S, or N atom. Heteroaryl groupsinclude monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings)systems. A ring-forming N atom in a heteroaryl group can also beoxidized to form an N-oxo moiety. Examples of heteroaryl groups includepyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl,pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl,isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl,indolinyl. In some cases, a heteroaryl group has from 1 to about 20carbon atoms, and in some cases from about 3 to 20 carbon atoms. In somecases, a heteroaryl group contains 3 to about 14, 3 to about 7, or 5-6ring-forming atoms. In some cases, a heteroaryl group has 1 to about 4,1 to about 3, or 1-2 heteroatoms.

A “heteroarylalkyl” group refers to an alkyl group substituted by aheteroaryl group. An example of a heteroarylalkyl group ispyridylmethyl.

The terms “halo” or “halogen” refer to a fluorine (F), chlorine (Cl),bromine (Br), or iodine (I) atom; preferably, F, Cl, or Br; morepreferably, F or Cl. The term “perhalogenated” refers to a moiety whereall hydrogens are replaced by halogens. The term “haloalkyl” refers toalkyl groups with a halogen replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. C₁-C₆ haloalkyl includes a straight chainor branched alkyl with six or fewer backbone carbon atoms and a halogenreplaces a hydrogen on one or more backbone carbons.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.C₁-C₆ alkoxy refers to moieties having six or fewer carbon atoms in thehydrocarbon backbone. Examples of alkoxy groups (or alkoxyl radicals)include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxygroups. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.Examples of substituted alkoxy groups include halogenated alkoxy groups.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “pharmaceutically acceptable salts” refers to derivatives ofcompounds that are modified by converting an existing acid or basemoiety to its salt form. Non-limiting examples of pharmaceuticallyacceptable salts include mineral or organic acid salts of basic residuessuch as amines; alkali or organic salts of acidic residues such ascarboxylic acids. Pharmaceutically acceptable salts include conventionalnon-toxic salts of a parent compound formed, for example, from non-toxicinorganic or organic acids. Pharmaceutically acceptable salts may besynthesized by conventional chemical methods from a parent compound thatcontains a basic or acidic moiety. Generally, such salts can be preparedby reacting a free acid or base form of these compounds with astoichiometric amount of an appropriate base or acid in water or anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts can be found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Journal of Pharmaceutical Science, 66, 2 (1977), andP. Stahl and C. Wermuth, editors, Handbook of Pharmaceutical Salts:Properties, Selection and Use, 2^(nd) Revised ed., Weinheim/Zürich:Wiley-VCH/VHCA (2011), each of which is incorporated herein by referencein its entirety.

Examples of suitable inorganic acids include hydrochloric acid,sulphuric acid, phosphoric acid, or hydrobromic acid, while examples ofsuitable organic acids include carboxylic acid, sulpho acid, orsulphonic acid, such as acetic acid, tartaric acid, lactic acid,propionic acid, glycolic acid, malonic acid, maleic acid, fumaric acid,tannic acid, succinic acid, alginic acid, benzoic acid, 2-phenoxybenzoicacid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid,maleic acid, salicylic acid, trifluoroacetic acid, 3-aminosalicylicacid, ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid,oxalic acid, gluconic acid, amino acids, methanesulphonic acid,ethanesulphonic acid, 2-hydroxyethanesulphonic acid,ethane-1,2-disulphonic acid, benzenesulphonic acid,4-methylbenzenesulphonic acid or naphthalene-2-sulphonic acid. Examplesof suitable inorganic bases include sodium hydroxide, potassiumhydroxide and ammonia, while examples of suitable organic bases includeamines, e.g., tertiary amines, such as trimethylamine, triethylamine,pyridine, N,N-dimethylaniline, quinoline, isoquinoline, α-picoline,β-picoline, γ-picoline, quinaldine, or pyrimidine.

The term “antibody” encompasses the structure that constitutes thenatural biological form of an antibody. In most mammals, includinghumans, and mice, this form is a tetramer and consists of two identicalpairs of two immunoglobulin chains, each pair having one light and oneheavy chain, each light chain comprising immunoglobulin domains V_(L)and C_(L), and each heavy chain comprising immunoglobulin domains V_(H),Cγ1, Cγ2, and Cγ3. In each pair, the light and heavy chain variableregions (V_(L) and V_(H)) are together responsible for binding to anantigen, and the constant regions (C_(L), Cγ1, Cγ2, and Cγ3,particularly Cγ2, and Cγ3) are responsible for antibody effectorfunctions. In some mammals, for example in camels and llamas,full-length antibodies may consist of only two heavy chains, each heavychain comprising immunoglobulin domains V_(H), Cγ2, and Cγ3. By“immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, includingfull-length antibodies, antibody fragments, and individualimmunoglobulin domains including V_(H), Cγ1, Cγ2, Cγ3, V_(L), and C_(L).

Based on the heavy-chain constant domain amino acid sequence, intactantibodies can be assigned to different “classes.” There are five-majorclasses (isotypes) of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into “subclasses,” e.g.,IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domainsthat correspond to the different antibody classes are called alpha,delta, epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known to one skilled in the art.

The terms “antibody” or “antigen-binding fragment,” respectively, referto intact molecules as well as functional fragments thereof, such asFab, a scFv-Fc bivalent molecule, F(ab′)₂, and Fv that can specificallyinteract with a desired target. In some cases, the antigen-bindingfragments comprise:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule, which can be        produced by digestion of whole antibody with the enzyme papain        to yield an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule that can be        obtained by treating whole antibody with pepsin, followed by        reduction, to yield an intact light chain and a portion of the        heavy chain; two Fab′ fragments are obtained per antibody        molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, a genetically engineered fragment containing the        variable region of the light chain and the variable region of        the heavy chain expressed as two chains;    -   (5) Single chain antibody (“SCA”), a genetically engineered        molecule containing the variable region of the light chain and        the variable region of the heavy chain, linked by a suitable        polypeptide linker as a genetically fused single chain molecule;        and    -   (6) scFv-Fc, is produced by fusing single-chain Fv (scFv) with a        hinge region from an immunoglobulin (Ig) such as an IgG, and Fc        regions.

In one embodiment, an antibody provided herein is a monoclonal antibody.In one embodiment, the antigen-binding fragment provided herein is asingle chain Fv (scFv), a diabody, a tandem scFv, a scFv-Fc bivalentmolecule, an Fab, Fab′, Fv, F(ab′)₂ or an antigen binding scaffold(e.g., affibody, monobody, anticalin, DARPin, Knottin).

The terms “binds,” “binding” or grammatical equivalents, refer tocompositions, directly or indirectly, having affinity for each other.“Specific binding” refers to selective binding between two molecules.For example, specific binding occurs between an antibody and an antigen.Typically, specific binding can be distinguished from non-specific whenthe dissociation constant (K_(D)) is less than about 1×10⁻⁵ M or lessthan about 1×10⁻⁶ M or 1×10⁻⁷ M. Specific binding can be detected, e.g.,by ELISA, immunoprecipitation, coprecipitation, with or without chemicalcrosslinking, and two-hybrid assays. Use of appropriate controls candistinguish between “specific” and “non-specific” binding. “Affinity” isthe strength of the binding interaction of two molecules, such as anantigen and its antibody, which is defined for antibodies and othermolecules with more than one binding site as the strength of binding ofthe ligand at one specified binding site. Although noncovalentattachment of a ligand to an antibody or other molecule is typically notas strong as a covalent attachment, a “high affinity” ligand binds to anantibody or other molecule with an affinity constant (K_(a)) greaterthan 10⁴ M⁻¹, typically 10⁵-10¹¹ M⁻¹; as determined by inhibition ELISAor an equivalent affinity determined by comparable techniques, such asScatchard plots or using K_(d)/dissociation constant, which is thereciprocal of the K_(a).

The term “selective” with respect to binding, inhibition, stimulation,or modulation means preferential binding, inhibition, stimulation, ormodulation, respectively, of a first activity relative to a secondactivity (e.g., preferential binding of one receptor to anotherreceptor; preferential inhibition relative to other receptors; orpreferential inhibition of a mutant to a wild-type or vice versa). Insome cases, binding is greater than two times, greater than five times,greater than ten times, greater than fifty times, greater than 100times, or greater than 1000 times more selective for the desiredmolecular target or pathway versus an undesired molecular target orpathway. In some cases, a compound will bind a first molecular target oraffect a pathway by at least 2-fold, at least 5-fold, at least 10-fold,at least 20-fold, at least 50-fold, at least 100-fold relative to asecond target or pathway under the same conditions. It will beappreciated that in preferred embodiments, binding to the D2-like familyof dopamine receptors or a member thereof, will be selective withrespect to the D1-like family of dopamine receptors or a member thereofby one of the foregoing amounts. The in vitro or in vivo activity of amolecular target or pathway may be measured by any suitable reproduciblemeans.

The term “modulating” refers to “stimulating” or “inhibiting” anactivity of a molecular target or pathway. For example, a compositionmodulates the activity of a molecular target or pathway if it stimulatesor inhibits that activity by at least 10%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 90%, at least about 95%, at least about 98%,or about 99% or more relative to the activity of that molecular targetor pathway under the same conditions but lacking the presence of thecomposition. In another example, a composition modulates the activity ofa molecular target or pathway if it stimulates or inhibits that activityby at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold,at least 50-fold, at least 100-fold relative to the activity of thattarget or pathway under the same conditions but lacking the presence ofthe composition. The activity of a molecular target or pathway may bemeasured by any reproducible means. For example, the activity of amolecular target or pathway may be measured in vitro or in vivo by asuitable assay known in the art for measuring the activity. Controlsamples (untreated with the composition) can be assigned a relativeactivity value of 100%.

In one embodiment, an antibody, antigen-binding fragment, or affinitytag binds its target with a K_(D) of 0.1 nM-10 mM, 0.1 nM-1 mM, orwithin the 0.1 nM range. In one embodiment, an antibody, antigen-bindingfragment, or affinity tag binds its target with a K_(D) of 0.1-2 nM,0.1-1 nM, 0.05-1 nM, 0.1-0.5 nM, or 0.1-0.2 nM. In one embodiment, anantibody, antigen-binding fragment, or affinity tag bind its targetdirectly. In one embodiment, an antibody, antigen-binding fragment, oraffinity tag bind its target indirectly, for example, binding as asecondary antibody that binds to an antibody bound to the target.

The word “label” refers to a compound or composition which is conjugatedor fused directly or indirectly to a reagent such as a nucleic acidprobe or an antibody and facilitates detection of the reagent to whichit is conjugated or fused. The label may itself be detectable (e.g.,radioisotopes or fluorescent labels) or, in the case of an enzymaticlabel, may catalyze chemical alteration of a substrate compound orcomposition, which is detectable.

The term “probe” refers to synthetic or biologically produced nucleicacids that contain specific nucleotide sequences which hybridize understringent conditions to target nucleic acid sequences. The terms“labeled probe,” “nucleic acid probe operably linked to a detectablelabel,” or “nucleic acid strand operably linked to a detectable label”refer to a probe which is prepared with a marker group or “detectablelabel” for detection. The marker group is attached at either the 5′ end,the 3′ end, internally, or a combination thereof. That is, one probe maybe attached to multiple markers. A preferred group is an identifyinglabel such as a fluorophore. A labeled probe may also comprise aplurality of different nucleic acid sequences each labeled with one ormore markers. Each marker may be the same or different. It may bebeneficial to label different probes (e.g., nucleic acid sequences) eachwith a different marker. This can be achieved by having a singledistinguishable group on each probe. For example, probe A is attached togroup X and probe B is attached to group Y. Alternatively, probe A isattached to groups X and Y while probe B is attached to groups Z and W.Alternatively, probe A is attached to groups X and Y, while probe B isattached to groups Y and Z. All probes “A” and “B” above would bedistinguishable and uniquely labeled.

By “tissue sample” is meant a collection of similar cells obtained fromtissue of a subject or patient, preferably containing nucleated cellswith chromosomal material. The four main human tissues are (1)epithelium; (2) connective tissues, including blood vessels, bone andcartilage; (3) muscle tissue; and (4) nerve tissue. A tissue samplesource may be solid tissue as from a fresh, frozen and/or preservedorgan or tissue sample or biopsy or aspirate; blood or a bloodconstituent; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid, or interstitial fluid; cells from a time ingestation or development of the subject. A tissue sample may be primaryor cultured cells or cell lines. A tissue sample may contain compoundsthat are not naturally intermixed with the tissue in nature such aspreservatives, anticoagulants, buffers, fixatives, nutrients, orantibiotics. By a tissue sample “section” is meant a single part orpiece of a tissue sample, e.g., a thin slice of tissue or cells cut froma tissue sample. Multiple sections of tissue samples may be taken andsubjected to analysis. A “cell line” refers to a permanently establishedcell culture that will proliferate given appropriate fresh medium andspace.

Detection Methods

In various aspects, provided herein are methods of detecting ormeasuring a target receptor (e.g., a dopamine receptor or a GPCR) in abiological sample. Targets are detected by contacting a sample with atarget detection reagent, e.g., an antibody or fragment thereof, and alabeling reagent. The presence or absence of targets are detected by thepresence or absence of the labeling reagent. In some cases, a sample iscontacted with the target detection and the labeling reagentsconcurrently e.g., the detection reagent is a primary antibody and thelabeling reagent is a fluorescent dye conjugated to it. Alternatively,the biological sample is contacted with the target detection andlabeling reagents sequentially, e.g., the detection reagent is a primaryantibody and the labeling reagent includes a secondary antibody. Forexample, a sample is incubated with a detection reagent, in some casestogether with a labeling reagent, under conditions that allow a complexbetween the detection reagent (and labeling reagent) and target to form.After complex formation the sample is optionally washed one or moretimes to remove unbound detection reagent (and labeling reagent). Whenthe sample is further contacted with a labeling reagent thatspecifically binds the detection reagent bound to the target, the samplecan optionally be washed one or more times to remove unbound labelingreagent. The presence or absence of the target in the sample is thendetermined by detecting the labeling reagent.

The methods described here provide for detecting multiple targets in asample. Multiple targets are identified by contacting the biologicalsample with additional detection reagents followed by additionallabeling reagent specific for the additional detection reagents usingthe methods described.

A detection moiety, i.e., detectable label, is a substance used tofacilitate identification and/or quantitation of a target. Detectionmoieties are directly observed or measured or indirectly observed ormeasured. Non-limiting examples of detection moieties includeradiolabels that can be measured with radiation-counting devices;pigments, dyes or other chromogens that can be visually observed ormeasured with a spectrophotometer; spin labels that can be measured witha spin label analyzer; and fluorescent moieties, where the output signalis generated by the excitation of a suitable molecular adduct and can bevisualized by excitation with light that is absorbed by the dye or canbe measured with standard fluorometers or imaging systems. A detectionmoiety can be a luminescent substance such as a phosphor or fluorogen; abioluminescent substance; a chemiluminescent substance, where the outputsignal is generated by chemical modification of the signal compound; ametal-containing substance; or an enzyme, where an enzyme-dependentsecondary generation of signal occurs, such as formation of a coloredproduct from a colorless substrate. The detection moiety may also takethe form of a chemical or biochemical, or an inert particle, includingcolloidal gold, microspheres, quantum dots, or inorganic crystals, suchas nanocrystals or phosphors. The term “detection moiety” or “detectablelabel” can also refer to a “tag” or hapten that can bind selectively toa labeled molecule, such that the labeled molecule, when addedsubsequently, is used to generate a detectable signal. For instance, onecan use biotin, iminobiotin or desthiobiotin as a tag and then use anavidin or streptavidin conjugate of horseradish peroxidase (HRP) to bindto the tag, and then use a chromogenic substrate (e.g.,tetramethylbenzidine) or a fluorogenic substrate, such as Amplex Red orAmplex Gold (Molecular Probes, Inc.) to detect the presence of HRP.Similarly, the tag can be a hapten or antigen (e.g., digoxigenin), andan enzymatically, fluorescently, or radioactively labeled antibody canbe used to bind to the tag. Numerous labels are known by those of skillin the art and non-limiting examples include particles, fluorescentdyes, haptens, enzymes and their chromogenic, fluorogenic, andchemiluminescent substrates.

A fluorophore is a chemical group that exhibits an absorption maximumbeyond 280 nm, and when covalently attached in a labeling reagentretains its spectral properties. Fluorophores include pyrene,anthracene, naphthalene, acridine, stilbene, indole or benzindole,oxazole or benzoxazole, thiazole or benzothiazole, porphyrin, cyanine,perylene, 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), carbocyanine,carbostyryl, salicylate, anthranilate, azulene, pyridine, quinoline,borapolyazaindacene, xanthene, oxazine or benzoxazine, carbazine,phenalenone, coumarin, benzofuran and benzphenalenone and derivativesthereof. Oxazines include resorufins, aminooxazinones, diaminooxazines,and their benzo-substituted analogs.

For a xanthene fluorophore, the fluorophore may be a fluorescein, arhodol, or a rhodamine. Fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, rhodol includes seminaphthorhodafluors. Alternatively, thefluorophore is a xanthene that is bound via a single covalent bond atthe 9-position of the xanthene. Preferred xanthenes include derivativesof 3H-xanthen-6-ol-3-one, 6-amino-3H-xanthen-3-one, or of6-amino-3H-xanthen-3-imine. Fluorophores include xanthene (rhodol,rhodamine, fluorescein and derivatives thereof) coumarin, cyanine,pyrene, oxazine and borapolyazaindacene. In addition, the fluorophorecan be sulfonated xanthenes, fluorinated xanthenes, sulfonatedcoumarins, fluorinated coumarins and sulfonated cyanines. The choice offluorophore in the labeling reagent will determine the absorption andfluorescence emission properties of the labeling reagent. Physicalproperties of a fluorophore label include spectral characteristics(absorption, emission and stokes shift), fluorescence intensity,lifetime, polarization and photo-bleaching rate can all be used todistinguish one fluorophore from another.

Typically, a fluorophore contains one or more aromatic or heteroaromaticrings that are optionally substituted by one or more substituents,including halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system,benzo, or other substituents typically found on fluorophores known inthe art.

Preferably, the detection moiety is a fluorescent dye. Fluorescent dyesinclude, for example, Fluorescein, Rhodamine, Texas Red, Cy2, Cy3, Cy5,Cy0, Cy0.5, Cy1, Cy1.5, Cy3.5, Cy7, VECTOR Red, ELF™ (Enzyme-LabeledFluorescence), FluorX, Calcein, Calcein-AM, CRYPTOFLUOR™'S, Orange (42kDa), Tangerine (35 kDa), Gold (31 kDa), Red (42 kDa), Crimson (40 kDa),BHMP, BHDMAP, Br-Oregon, Lucifer Yellow, Alexa dye family,N-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl) (NBD), BODIPY™,boron dipyrromethene difluoride, Oregon Green, MITOTRACKER™ Red, DiOC7(3), DilC18, Phycoerythrin, Phycobiliproteins BPE (240 kDa) RPE (240kDa) CPC (264 kDa) APC (104 kDa), Spectrum Blue, Spectrum Aqua, SpectrumGreen, Spectrum Gold, Spectrum Orange, Spectrum Red, NADH, NADPH, FAD,Infra-Red (IR) Dyes, Cyclic GDP-Ribose (cGDPR), Calcofluor White,Tyrosine and Tryptophan. Many fluorophores can also function aschromophores and thus they are also preferred chromophores.

In addition to fluorophores, enzymes also find use as detectablemoieties. Enzymes are desirable detectable moieties becauseamplification of a detectable signal can be achieved resulting inincreased assay sensitivity. The enzyme itself does not produce adetectable response but breaks down a substrate when it is contacted byan appropriate substrate such that the converted substrate produces afluorescent, colorimetric or luminescent signal. Enzymes amplify adetectable signal because one enzyme on a labeling reagent can result inmultiple substrates being converted to a detectable signal. This isadvantageous where there is a low quantity of target present in thesample or a fluorophore does not exist that will give comparable orstronger signal than the enzyme. However, fluorophores are preferredbecause they do not require additional assay steps, and thus reduce theoverall time to complete an assay. The enzyme substrate is selected toyield the preferred measurable product, e.g. colorimetric, fluorescentor chemiluminescence. Such substrates are extensively used in the art.

A preferred colorimetric or fluorogenic substrate and enzyme combinationuses oxidoreductases, such as horseradish peroxidase and a substratesuch as 3,3′-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazol-e (AEC),which yield a distinguishing color (brown and red, respectively). Othernon-limiting examples of colorimetric oxidoreductase substrates thatyield detectable products include:2,2-azino-bis(3-ethylbenzothiaz-oline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Non-limitingexamples of fluorogenic substrates include: homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplexe Red reagent and its variants andreduced dihydroxanthenes, including dihydrofluoresceins anddihydrorhodamines including dihydrorhodamine 123. Peroxidase substratesthat are tyramides represent a class of peroxidase substrates that canbe intrinsically detectable before action of the enzyme but are “fixedin place” by the action of a peroxidase in the process described astyramide signal amplification (TSA). These substrates are extensivelyutilized to label targets in samples that are cells, tissues or arraysfor their subsequent detection by microscopy, flow cytometry, opticalscanning and fluorometry.

Additional colorimetric (and in some cases fluorogenic) substrate andenzyme combinations use a phosphatase enzyme such as an acidphosphatase, an alkaline phosphatase or a recombinant version of such aphosphatase in combination with a colorimetric substrate such as5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy4-methylcoumarinyl phosphate (DiFMUP) fluoresceindiphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate,9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAOphosphate), or ELF 97, ELF 39 or related phosphates.

Glycosidases, in particular β-galactosidase, β-glucuronidase andβ-glucosidase, are additional suitable enzymes. Non-limiting examples ofappropriate colorimetric substrates include: 5-bromo4-chloro-3-indolylβ-D-galactopyranoside (X-gal) and similar indolyl galactosides,glucosides, and glucuronides, o-nitrophenyl β-galactopyranoside (ONPG)and p-nitrophenyl β-D-galactopyranoside. Preferred fluorogenicsubstrates include resorufin β-D-galactopyranoside, fluoresceindigalactoside (FDG), fluorescein diglucuronide and their structuralvariants, 4-methylumbelliferyl β-D-galactopyranoside,carboxyumbelliferyl β-D-galactopyranoside and fluorinated coumarinβ-D-galactopyranosides. Additional enzymes include hydrolases, such ascholinesterases and peptidases; oxidases, such as glucose oxidase; andcytochrome oxidases and reductases for which suitable substrates areknown.

Enzymes and their substrates that produce chemiluminescence arepreferred for some assays. These include, for example, natural andrecombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful. For example,the enzyme is luciferase or aequorin, and the substrates are luciferine,ATP, Ca⁺⁺ and coelenterazine.

In addition to enzymes, haptens such as biotin are useful detectablemoieties. Biotin is useful as it is in an enzyme system that can furtheramplify a detectable signal, and it can serve as a tag in affinitychromatography for isolation purposes. For detection, an enzymeconjugate that has affinity for biotin is used, such as avidin-HRP.Subsequently, a peroxidase substrate is added to produce a detectablesignal. Haptens also include hormones, naturally occurring and syntheticdrugs, pollutants, allergens, affector molecules, growth factors,chemokines, cytokines, lymphokines, amino acids, peptides, chemicalintermediates, or nucleotides.

In some cases, a detectable moiety is a fluorescent protein. Exemplaryfluorescent proteins include green fluorescent protein (GFP),phycobiliproteins and their derivatives, luciferase or aequorin.Fluorescent proteins, especially phycobiliprotein, are particularlyuseful for creating tandem dye labeled labeling reagents. These tandemdyes comprise a fluorescent protein and a fluorophore to obtain a largerstokes shift where the emission spectra is farther shifted from thefluorescent protein's absorption spectra. This is particularlyadvantageous to detect a low amount of target in a sample where theemitted fluorescent light is maximally optimized, in other words thefluorescent protein reabsorbs little to none of the emitted light. Thefluorescent protein and fluorophore function as an energy transfer pairwhere the fluorescent protein emits at a wavelength the fluorophoreabsorbs, and the fluorphore then emits at a wavelength farther from thefluorescent protein than could be obtained with only the fluorescentprotein. A particularly useful combination is phycobiliproteins andsulforhodamine fluorophores, or sulfonated cyanine fluorophores; orsulfonated xanthene derivatives. Alternatively, the fluorophore is anenergy donor and the fluorescent protein is an energy acceptor.

Methods of Visualizing the Detection Moiety Depend on the Label.

In some cases, a sample is illuminated with a light wavelength selectedto give a detectable optical response, and observed with a means fordetecting that response. Equipment useful for illuminating fluorescentcompounds include hand-held ultraviolet lamps, mercury arc lamps, xenonlamps, lasers and laser diodes. These illumination sources are opticallyintegrated into laser scanners, fluorescent microplate readers orstandard or microfluorometers. The degree or location of signal,compared to a standard or expected response, indicates whether and towhat degree the sample possesses a given characteristic or desiredtarget.

An optical response is detected by visual inspection, or by using one ofthe following devices: CCD camera, video camera, photographic film,laser-scanning devices, fluorometers, photodiodes, quantum counters,epifluorescence microscopes, scanning microscopes, flow cytometers,fluorescence microplate readers, or by means for amplifying the signalsuch as photomultiplier tubes. When a sample is examined using a flowcytometer, examination of it optionally includes sorting portions of itaccording to their fluorescence response.

When an indirectly detectable label is used, then illuminating typicallyincludes adding a reagent to produce a detectable signal such as acolorimetric enzyme substrate. Radioisotopes are also consideredindirectly detectable where an additional reagent is not needed, ratherthe radioisotope is exposed to X-ray film or other mechanism to recordand measure the signal. This is true for some chemiluminescent signalsthat are observed after exposure to film.

I. ONC201 (Compound (1)), Salts Thereof and Syntheses Thereof

ONC201 (compound (1))

and its analogs, and their pharmaceutically acceptable salts, as well assyntheses for them, are provided herein. In in vitro models, animalmodels, and human clinical trials, ONC201 has broad anti-canceractivity, low toxicity including few, if any, adverse effects, lowgenotoxicity, and high bioavailability including orally. These featuresallow ONC 201 and various analogs to be well suited for a variety ofapplications. ONC201 can be synthesized as shown in Scheme 1.

Synthesis of an ONC201 dihydrochloride salt starts with commerciallyavailable intermediary N-Benzyl-3-carbomethoxy-4-piperidonehydrochloride, compound (3). In some embodiments, the synthesis includesneutralizing compound (3) with a base (Step 1) to produce compound (4),a free base. For example, compound (3) is neutralized with an inorganicbase to produce compound (4). Alternatively, compound (3) is neutralizedwith an organic base to produce compound (4). In some embodiments,compound (3) is neutralized in the presence of an alcohol, for example,n-butanol. In some embodiments, compound (3) is neutralized in thepresence of at least one organic solvent (e.g., n-butanol, ethyl acetateor both). In some embodiments, compound (3) is neutralized in thepresence of a base and at least one organic solvent (e.g., NaHCO₃ andn-butanol). In one embodiment, compound (3) is neutralized in thepresence of n-butanol and triethyl amine (Et₃N).

In some embodiments, the synthesis includes reacting compounds (4) with(5) (Step 2) to produce intermediary compound (1). In some embodiments,Step 2 includes heating compounds (4) with (5). In some embodiments,Step 2 includes refluxing heating compounds (4) with (5) in the presenceof a solvent. In some embodiments, Step 2 includes use of Dean-starktrap to remove water and/or methanol (MeOH) formed in the reaction.

In some embodiments, an ONC201 dihydrochloride salt is synthesized (Step3). In some embodiments, Step 3 includes treating ONC201 with HCl indioxane. In some embodiments, Step 3 includes treating ONC201 with 4NHCl in dioxane. In some embodiments, the synthesis optionally includesrecrystallizing the ONC201 di-salt. Preferrably, the ONC201di-hydrochloride salt is synthesized as shown in Scheme 2.

II. TNF-Related Apoptosis-Inducing Ligand (“Trail”)

TRAIL protein can be assayed in a sample obtained from a subject todetect TRAIL expression induced by compounds and their salts describedherein. Immunoassays can be used to assay TRAIL, including enzyme-linkedimmunosorbent assay (ELISA), enzyme-linked immunofiltration assay(ELIFA), flow cytometry, immunoblot, immunoprecipitation,immunohistochemistry, immunocytochemistry, luminescent immunoassay(LIA), fluorescent immunoassay (FIA), and radioimmunoassay. Qualitativeand/or quantitative results may be obtained. Suitable methods forqualitative and quantitative assays are described in standardreferences, including Harlow & Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1988; Breitling & Diibel,Recombinant Antibodies, John Wiley & Sons, New York, 1999; H. Zola,Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies andEngineered Antibody Derivatives, Basics: From Background to Bench, BIOSScientific Publishers, 2000; B. K. C. Lo, Antibody Engineering: Methodsand Protocols, Methods in Molecular Biology, Humana Press, 2003; Ausubelet al., Eds., Short Protocols in Molecular Biology, Current Protocols,Wiley, 2002; S. Klussman, Ed., The Aptamer Handbook: FunctionalOligonucleotides and Their Applications, Wiley, 2006; Ormerod, M. G.,Flow Cytometry: a practical approach, Oxford University Press, 2000;Givan, A. L., Flow Cytometry: first principles, Wiley, New York, 2001;Gorczyca, W., Flow Cytometry in Neoplastic Hematology:morphologic-immunophenotypic correlation, Taylor & Francis, 2006;Crowther, J. R., The ELISA Guidebook (Methods in Molecular Biology),Humana Press, 2000; Wild, D., The Immunoassay Handbook, 3^(rd) Edition,Elsevier Science, 2005; and Sambrook & Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 3^(rd) ed.,2001.

Assays to analyze a sample for TRAIL to detect an effect of apharmaceutical composition are described in U.S. Pat. No. 8,673,923,incorporated by reference herein in its entirety.

In some embodiments, TRAIL assays are used to monitor a subject. Forexample, a sample is obtained from a subject before treatment with apharmaceutical agent and at one or more times during and/or followingtreatment to assess treatment effectiveness. In another example, asample is obtained from a subject at various times to assess the courseor progress of disease or healing. In one embodiment, death receptorsfrom circulating tumor cells are assayed to see if a treatment describedhere increases the amount or type of death receptors.

Cancers treated using methods and compositions described herein arecharacterized by abnormal cell proliferation including pre-neoplastichyperproliferation, cancer in-situ, neoplasms and metastasis. Methodsand compositions described herein can be used for prophylaxis, as wellas amelioration of cancer signs or symptoms. Cancer“treatment” in asubject includes: preventing, inhibiting or ameliorating cancer in thesubject, such as slowing cancer progression or reducing or amelioratinga cancer sign or symptom. Examples of cancers treated using methods andcompositions described herein include breast cancer, CNS cancers, coloncancer, ovarian cancer, prostate cancer, leukemia, lung cancer, andlymphoma.

III. Compounds of Formula (10) and Salts Thereof

In one aspect, provided herein are compounds and salts of formula (10)and methods of making them. Those skilled in the art will understandthat the general principles and concepts described here in conjunctionwith ONC201 (compound (1)) and its salts, including principles andconcepts related to methods and pharmaceutical compositions, apply withequal force to compounds of formula (10) and salts thereof.

In one embodiment, provided herein are compounds of formula (10):

wherein R₁ and R₂ are independently selected from H, alkyl, aryl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl,aralkoxy, aralkylthio, and acyl radicals. In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph) (ONC201). In one embodiment, R₁ is CH₂Ph and R₂is CH₂-(2,4-di F-Ph) (ONC206). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph) (ONC212). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(3,4-di F-Ph) (ONC213). In one embodiment, R₁ is CH₂-(3,4-di-Cl-Ph)and R₂ is CH₂-(4-CF₃-Ph) (ONC234). In one embodiment, R₁ isCH₂-3-thienyl and R₂ is CH₂-(4-CF₃-Ph) (ONC236).

In one embodiment, R₁ and R₂ are independently selected from H,C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone,C₁₋₄benzyl-piperazine, C₁₋₄alkylthienyl, C₁₋₄alkylpyridinyl,C₁₋₄alkylisoxazolidinyl, C₁₋₄alkylmorpholinyl, C₁₋₄alkylthiazolyl, andC₁₋₄alkylpyrazinyl wherein C₁₋₄alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, C₁₋₄alkylthienyl,C₁₋₄alkylpyridinyl, C₁₋₄alkylisoxazolidinyl, C₁₋₄alkylthiazolyl,C₁₋₄alkylmorpholinyl, and C₁₋₄alkylpyrazinyl are optionally substitutedwith C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, orhalo. In one embodiment, R₁ and/or R₂ is a substituted or unsubstituted,arylalkyl or heteroarylalkyl. In one embodiment, the heteroarylalkyl isselected from C₁₋₄alkylpyrrolyl, C₁₋₄alkylfuryl, C₁₋₄alkylpyridyl,C₁₋₄alkyl-1,2,4-thiadiazolyl, C₁₋₄alkylthienyl, C₁₋₄alkylisothiazolyl,C₁₋₄alkylimidazolyl, C₁₋₄alkyltetrazolyl, C₁₋₄alkylpyrazinyl,C₁₋₄alkylpyrimidyl, C₁₋₄alkylquinolyl, C₁₋₄alkylpyrazolyl,C₁₋₄alkylisoquinolyl, C₁₋₄alkylthiophenyl, C₁₋₄alkylbenzothienyl,C₁₋₄alkylisobenzofuryl, C₁₋₄alkylindolyl, C₁₋₄alkylpurinyl,C₁₋₄alkylcarbazolyl, C₁₋₄alkylbenzimidazolyl, and C₁₋₄alkylisoxazolyl.

In one embodiment, R₁ and/or R₂ is a benzyl optionally substituted withone or more of the following substituents on the benzyl ring: X, —CH₃,—NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂-C₄ alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1), —OC_(p)X_(2p+1),OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m),C(O)R^(m), and C(O)OR^(m); R^(m) and R^(n) are independently selectedfrom H or a C₁-C₄ alkyl; and where p is an integer from 2 to 20 and X ishalogen, including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl.

In one embodiment, R₁ is selected from H, CH₃, CH₂Ph, CH₂-(4-CF₃-Ph),CH₂-(4-F-Ph), CH₂-(4-Cl-Ph), CH₂—(OCH₃-Ph), CH₂-((2-Cl)-Ph),CH₂-(2-thienyl), CH₂-(3-thienyl), CH₂-2-pyridinyl,CH₂-4-methyl-2-thiazolyl, CH₂-2-pyrazinyl, CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-(3,4-di Cl-Ph),CH₂-(3,4-di F-Ph), CH₂-(3,5-di F-Ph), CH₂-((2-CH₃)-Ph), CH₂CH(OH)Ph,(4-F-Ph)-4-oxobutyl, CH₂CH₂NHCOOC(CH₃)₃, CH₂CH₂CH₂NH₂, and CD₂C₆D₅. Inone embodiment, R₂ is selected from H, CH₃, CH₂Ph, CH₂-(4-CF₃-Ph),CH₂-((2-Cl)-Ph), CH₂-((2-F)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-(2,4-di Cl-Ph),CH₂-(3,4-di Cl-Ph), CH₂-(3,4-di F-Ph), CH₂-(3,5-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂(2-CH₃, 4-F-Ph), CH₂-((4-OCH₃)-Ph),CH₂-(3-pyridinyl), CH₂-(3-isoxazolidinyl), CH₂CH₂-(4-morpholinyl),CH₂-(2-F, 4-CF₃-Ph), CH₂CH(OH)Ph, (CH₂)₃CO-4F-Ph, (4-F-Ph)-4-oxobutyl,CH₂CH₂NHCOOC(CH₃)₃, CH₂CH₂CH₂NH₂, and CD₂C₆D₅.

In one embodiment, R₁ is H. In one embodiment, R₁ is an unsubstituted orsubstituted arylalkyl, e.g., a benzyl (CH₂Ph) or phenylethyl (CH₂CH₂Ph)group. In one embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,e.g., benzyl or phenylethyl. In one embodiment, the arylalkyl issubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from halo, CH₃, CF₃ or OC_(H3). In oneembodiment, R₂ is a substituted or an unsubstitutedheterocycloalkylalkyl, e.g., piperazinylalkyl or morpholinoalkyl. In oneembodiment, R₂ is a substituted or an unsubstituted heteroarylalkyl,e.g., pyridylmethyl or isoxazolidinylmethyl. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo. In oneembodiment, the heterocycloalkylalkyl or heteroarylalkyl is substitutedwith at least one substituent selected from halo, CH₃, CF₃ or OCH₃.

In one embodiment, compound (10) has the structure of formula (80):

wherein R_(a1), R_(a2), R_(a3), R_(a4), R_(a5), R_(b1), R_(b2), R_(b3),R_(b4), and R_(b5) are each independently selected from the groupconsisting of H, X, —CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂-C₄ alkyl,—CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃,—OC_(p)H_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n),NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) andR^(n) are independently selected from H or a C₁-C₄ alkyl; and where p isan integer from 2 to 20 and X is a halogen.

In one embodiment, compound (10) has the structure of formula (90)

wherein R₂ is as defined above, and wherein R_(b1), R_(b2), R_(b3),R_(b4), and R_(b5) are each independently selected from the groupconsisting of H, X, —CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂₋₄ alkyl,—CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃,—OC_(p)H_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n),NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) andR^(n) are independently selected from H or a C₁₋₄alkyl; and where p isan integer from 2 to 20 and X is a halogen.

In one embodiment, compound (10) has the structure of formula (40)

where R₁ is as defined above, and where R_(a1), R_(a2), R_(a3), R_(a4),and R_(a5) are each independently selected from H, X, —CH₃, —NO₂, —OCH₃,—CN, —CXH₂, —CX₂H, C₂₋₄alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m),NR^(m)R^(n), NR^(m)C(O)R^(o), SOR^(m), SO₂R^(m), C(O)R^(m), andC(O)OR^(m); R^(m) and R^(o) are independently selected from H or a C₁₋₄alkyl; p is an integer from 2 to 20; and X is a halogen. In oneembodiment, R₁ is H. In one embodiment, R₁ is a substituted orunsubstituted arylalkyl, such as benzyl or phenylethyl. In oneembodiment, the arylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl,hydroxyl, perhalogenated C₁₋₄alkyl, or halo. In one embodiment, thebenzyl is substituted with one or more halo. In one embodiment, thebenzyl is substituted with one or more substituents selected from halo,CH₃, CF₃, and OCH₃. In one embodiment, the benzyl is substituted withone halo, e.g., F at an ortho or para position. In one embodiment, thebenzyl is substituted with two halogen, e.g., F at both meta positions.

In one embodiment, compound (40) has the structure of compound (45):

where R_(a1), R_(a2), R_(a3), R_(a4), and R_(a5) are as defined above.In one embodiment, the benzyl is substituted with one or more halogens.In one embodiment, the benzyl is substituted with one or moresubstituents selected from halo, CH₃, CF₃, and OCH₃. In one embodiment,R_(a1) or R_(a5) is a halo, e.g., F. In one embodiment, both R_(a2) andR_(a3) are halo, e.g., F.

In one embodiment, compound (10) has the structure of compound (50)

wherein R₁ is as defined above, and wherein R_(b) is selected from H, X,—CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂₋₄alkyl, —CX₃, —CH₂(CX₃),—CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1),—OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m),SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) and R^(o) are independentlyselected from H or C₁₋₄alkyl; and where p is an integer from 2 to 20 andX is a halogen, and wherein R_(a1), R_(a2), R_(a4), and R_(a5) are eachindependently selected from H, X, —CH₃, —NO₂, —OCH₃, —CN, —CXH₂, —CX₂H,C₂₋₄alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃,—OC_(p)H_(2p+1), —OC_(p)X_(2p+1), OR^(m), SR^(m), NR^(m)R^(o),NR^(m)C(O)R^(o), SOR^(m), SO₂R^(m), C(O)R^(m), and C(O)OR^(m); R^(m) andR^(n) are independently selected from H or C₁₋₄ alkyl; and where p is aninteger from 2 to 20 and X is a halogen. In one embodiment, R₁ is H. Inone embodiment, R₁ is a substituted or unsubstituted arylalkyl, such asa benzyl or phenylethyl group. In one embodiment, the arylalkyl issubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, R_(b) is selected from halo, CH₃,CF₃, and OCH₃. In one embodiment, one or more of R_(a1), R_(a2), R_(a4),and R_(a5) is selected from halo, CH₃, CF₃, and OCH₃. In one embodiment,R_(a1), R_(a2), R_(a4), and R_(a5) are H, and R_(b) is selected fromhalo, CH₃, CF₃, and OCH₃. In one embodiment, R_(b) is halogen, e.g., F,and R_(a1) is CH₃. In one embodiment, R_(b) is F or Cl, and R_(a2) is For Cl. In one embodiment, R_(b) is CF₃. In one embodiment, R_(b) isOCH₃. In one embodiment, R_(b) and R_(a1) are Cl.

In one embodiment, compound (50) has the structure of compound (55):

where R_(a1), R_(a2), R_(a4), R_(a5), and R_(b) are as defined above. Inone embodiment, R_(b) is selected from halo, CH₃, CF₃, and OCH₃. In oneembodiment, one or more of R_(a1), R_(a2), R_(a4), and R_(a5) isselected from halo, CH₃, CF₃, and OCH₃. In one embodiment, R_(a1),R_(a2), R_(a4), and R_(a5) are H, and R_(b) is selected from halo, CH₃,CF₃, and OCH₃. In one embodiment, R_(b) is halo, e.g., F, and R_(a1) isCH₃. In one embodiment, R_(b) is F or Cl, and R_(a2) is F or Cl. In oneembodiment, R_(b) is CF₃. In one embodiment, R_(b) is OCH₃. In oneembodiment, R_(b) and R_(a1) are Cl.

In one embodiment, compound (10) has the structure of compound (60)

In one embodiment, R₁ is H. In one embodiment, R₁ is a substituted orunsubstituted arylalkyl, such as benzyl or phenylethyl. In oneembodiment, R₁ is a substituted or unsubstituted heterocycloalkylalkylor a substituted or unsubstituted heteroarylalkyl, such asCH₂-(2-thienyl), CH₂-(3-thienyl), CH₂-4-methyl-2-thiazolyl,CH₂-2-pyrazinyl, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(3-isoxazolidinyl),CH₂-2-pyridinyl, CH₂-3-pyridinyl, and CH₂CH₂-(4-morpholinyl). In oneembodiment, the arylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl,hydroxyl, perhalogenated C₁₋₄alkyl, or halo. In one embodiment, thebenzyl is substituted with one or more halogens. In one embodiment, thebenzyl is substituted with one or more substituents selected from halo(e.g., F), CH₃, CF₃, and OCH₃. In one embodiment, the benzyl issubstituted at the para position with a halo, CH₃, CF₃, or OCH₃substituent. In one embodiment, R₁ is fluorophenyloxobutyl orhydroxyphenylethyl.

Compounds of formula (10) (imipridones) are synthesized starting from asubstituted piperidone, which is converted by reaction with asubstituted aminoimidazoline to give the core compound (10). There aretwo routes, one in which the R₁ substituent is present in the piperidone(e.g., 68). In that route, (68) is acylated with dimethyl carbonateusing sodium hydride in toluene at 80° C. to form piperidone ester (69).Commercially available methylthioimidazoline HI salt (63) is reactedwith an amine in dioxane at 70° C. to afford the R₂-substitutedaminoimidazoline (64) as its HI salt. Direct reaction of (64) withpiperidone ester (69) in 1-butanol at reflux with removal of water via aDean-Stark trap over 3-6 h gives the tricylic compound (10). In avariant of this scheme, N—BOC protected piperidone (61) is converted bythe same methods to BOC protected compound (65), which is treated withHCl in dioxane to remove the BOC group and then converted to the freebase of (66) with 1N NaOH with extraction with methylene chloride.Subsequent treatment of (66) with a halide (67) or epoxide (70) affordsdesired compound (10).

Crude products may be purified by column chromatography eluting withmethylene chloride:methanol or by HPLC using acetonitrile:TFA:H₂O toproduce either free bases or TFA salts as final products. Treatment offree bases with HCl in dioxane or lyophilization of TFA salts generatesproducts (10) as HCl or TFA salts. Alternatively, the free base may betreated with another inorganic or organic acid to form other salts,generally selected from those known to be pharmaceutically acceptable.Salts of compound (10) are usually solids and examples have beencrystallized from ethanol or other solvents to give high qualitycrystals. The tricyclic structure of compound (1) has been definitivelyconfirmed by an X-ray crystal structure and NMR.

Compounds described herein can be used, with or without an aminoalkyllinker (e.g., compound (33)), to identify molecules (e.g., proteins)that interact with them in a cellular context. Expression of thesebinding targets may be used to predict response to imipridones oranalogs thereof (i.e. serve as biomarkers). These compounds can also beused to screen for structurally unrelated molecules using competitionassays known in the art to identify drugs able to outcompete the targetinteraction with a higher affinity. In addition, these molecules mayhave improved drug properties or allow additional applications byaltering drug properties including safety, potency, pharmacokinetics,biodistribution, or metabolism.

TABLE 1 EXAMPLES OF COMPOUNDS OF FORMULA (10) ONC No. Number R₁ R₂  1ONC201 CH₂Ph CH₂—((2-CH₃)—Ph) 13 CH₂Ph CH₃ 14 ONC202 CH₂PhCH₂—((2-Cl)—Ph) 15 ONC203 CH₂Ph CH₂—(2-thienyl) 16 ONC204 CH₂Ph CH₂CH₂Ph17 ONC205 CH₂Ph CH₂CH₂(4-N-benzyl- piperazine) 18 ONC206 CH₂PhCH₂—(2,4-di F—Ph) 19 ONC207 H CH₂—((2-CH₃)—Ph) 20 ONC208 CH₃CH₂—((2-CH₃)—Ph) 21 ONC209 CH₂CH₂Ph CH₂—((2-CH₃)—Ph) 22 CH₂CH₂—(4-N-CH₂—((2-CH₃)—Ph) benzyl-piperizine) 23 CH₂CHOHPh CH₂—((2-CH₃)—Ph) 24(CH₂)₃CO-4F—Ph CH₂—((2-CH₃)—Ph) 32 ONC215 CH₂CH₂NHCOOC(CH₃)₃CH₂—((2-CH₃)—Ph) 33 ONC216 CH₂CH₂CH₂NH₂ CH₂—((2-CH₃)—Ph) 41 ONC210 CH₂PhCH₂—(3,5-di F—Ph) 51 ONC211 CH₂Ph CH₂—(3,4-di Cl—Ph) 52 ONC212 CH₂PhCH₂—(4-CF₃—Ph) 53 ONC213 CH₂Ph CH₂—(3,4-di F—Ph) 54 ONC214 CD₂C₆D₅CH₂—((2-CH₃)—Ph) 43 ONC217 CH₂Ph CH₂—(2-F-Ph) 55 ONC218 CH₂Ph CH₂(2-CH₃,4-F—Ph) 56 ONC219 CH₂Ph CH₂—(2,4-di Cl-Ph) 57 ONC220 CH₂PhCH₂—((4-OCH₃)—Ph) 34 ONC226 CH₂Ph CH₂—(3-pyridinyl) 35 ONC222 CH₂PhCH₂—(3- isoxazoliclinyl) 36 ONC224 CH₂Ph CH₂CH₂—(4- morpholinyl) 37ONC223 CH₂Ph CH₂—(4-CH₃—Ph) 38 ONC221 H CH₂—(4-CF₃—Ph) 73 ONC227CH₂—(4-CF₃—Ph) CH₂—(4-CF₃—Ph) 72 ONC225 CH₂Ph CH₂—(2-F, 4-CF₃—Ph) 74ONC228 CH₂—(4-F—Ph) CH₂—(4-CF₃—Ph) 75 ONC229 CH₂—(OCH₃—Ph)CH₂—(4-CF₃—Ph) 76 ONC230 (4-F—Ph)-4-oxobutyl CH₂—(4-CF₃—Ph) 77 ONC231CH₂—3-pyridyl CH₂—(4-CF₃—Ph) 78 ONC232 CH₂—4-methyl-2- CH₂—(4-CF₃—Ph)thiazolyl 79 ONC233 CH₂—2-pyrazinyl CH₂—(4-CF₃—Ph) 81 ONC234 CH₂—(3,4-diCl—Ph) CH₂—(4-CF₃—Ph) 82 ONC235 CH₂—(4-Cl—Ph) CH₂—(4-CF₃—Ph) 83 ONC236CH₂—3-thienyl CH₂—(4-CF₃—Ph) 84 ONC237 CH₂CH(OH)Ph CH₂—(4-CF₃—Ph)

IV. Assessing Sensitivity and Efficacy of Treatment Regimens

Measuring expression, gene mutation, or gene copy number of a dopaminereceptor or other G protein-coupled receptor (GPCR) may be used topredict response or sensitivity to methods of treatment described hereinor to identify subjects likely to be responsive to methods of treatmentdescribed herein, such as treatment with a compound of formula (10), apharmaceutically acceptable salt thereof, or an analog thereof. In oneaspect, provided herein are methods of identifying whether a subjecthaving a condition is likely to be responsive to a treatment regimendescribed herein. In one embodiment, the methods comprises (i) obtaininga biological sample from the subject; (ii) measuring expression levelsof at least one dopamine receptor or GPCR in the sample; (iii) comparingthe levels measured in the sample to those for a pre-determinedstandard; and (iv) determining whether the subject is likely to beresponsive to the treatment regimen, based on the levels measured in thesample to those for the pre-determined standard. In one embodiment, thestep of measuring an expression level in the sample include the steps of(i) contacting the sample with an antibody or antigen-binding fragmentthat specifically binds to the receptor to form a complex of theantibody or antigen-binding fragment with the receptor; and (ii)measuring the amount of the complex. In one embodiment, the subject has,or is at risk of having, cancer. In one embodiment, the cancer is aneuro-oncology disease. In one embodiment, the cancer is aneuroendocrine tumor. In one embodiment, the cancer is selected from thegroup consisting of meningioma, ependymoma, glioma, neuroblastoma, anddiffuse intrinsic pontine glioma. In one embodiment, the subject has, oris at risk of having, a psychiatric disorder. For example, thepsychiatric disorder is selected from psychosis, bipolar disorder, andmajor depressive disorder. In one embodiment, the subject has, or is atrisk of having, an infection, such as a bacterial infection. In oneembodiment, the infection is a gram-positive bacterial infection. In oneembodiment, the infection is a gram-negative bacterial infection. In oneembodiment, the infection is an infection of a bacteria selected fromEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspecies. In one embodiment, the gram-positive bacterial infection is aStaphylococcus infection. For example, the Staphylococcus infection isan S. aureus infection (e.g., a methicillin-resistant S. aureus (MRSA)infection). In one embodiment, the treatment regimen comprisesadministering an effective amount of a therapeutic, such as a compoundof formula (10), a pharmaceutically acceptable salt thereof, or ananalog thereof. In one embodiment, the dopamine receptor is from theD2-like family. In one embodiment, the dopamine receptor is DRD2, DRD3,or both. In one embodiment, the dopamine receptor is DRD4. In oneembodiment, the GPCR is a Class A GPCR. In one embodiment, the GPCR isGPR132. In one embodiment, the GPCR is selected from GPR132, GPR91,MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, and combinationsthereof. In one embodiment, the dopamine receptor is DRD5, the treatmentregimen comprises administering an effective amount of a therapeutic,such as a compound of formula (10) or a pharmaceutically acceptable saltthereof, and an increased DRD5 expression level measured in the samplerelative to the pre-determined standard indicates that the subject is oris not likely to be responsive to the treatment regimen.

In another aspect, provided herein are methods of assessing theeffectiveness of a treatment regimen described herein, monitoring, orproviding a prognosis for a subject with a condition. In one embodiment,the methods comprises (i) obtaining a biological sample from thesubject; (ii) measuring expression levels of at least one dopaminereceptor or GPCR in the sample; (iii) comparing the levels measured inthe sample to those for a pre-determined standard; and (iv) determininga prognosis or whether the subject is responsive to the treatmentregimen, based on the levels measured in the sample to those for thepre-determined standard. In one embodiment, the step of measuring anexpression level of a dopamine receptor or GPCR in the sample includethe steps of (i) contacting the sample with an antibody orantigen-binding fragment that specifically binds to the receptor to forma complex of the antibody or antigen-binding fragment with the receptor;and (ii) measuring the amount of the complex. In one embodiment, themethods comprise (i) obtaining a biological sample from the subject;(ii) measuring gene copy number or mutations in at least one dopaminereceptor in the sample; (iii) comparing the copy number measured ormutations found in the sample to those for a pre-determined standard;and (iv) determining whether the subject is responsive to the treatmentregimen, based on the copy number measured or mutations found in thesample to those for the pre-determined standard. In one embodiment, thesubject has, or is at risk of having, cancer. In one embodiment, thecancer is a neuro-oncology disease. In one embodiment, the cancer is aneuroendocrine tumor. In one embodiment, the cancer is selected from thegroup consisting of meningioma, ependymoma, glioma, neuroblastoma, anddiffuse intrinsic pontine glioma. In one embodiment, the subject has, oris at risk of having, a psychiatric disorder. For example, thepsychiatric disorder is selected from psychosis, bipolar disorder, andmajor depressive disorder. In one embodiment, the subject has, or is atrisk of having, an infection, such as a bacterial infection. In oneembodiment, the infection is a gram-negative bacterial infection. In oneembodiment, the infection is a gram-positive bacterial infection. In oneembodiment, the infection is an infection of a bacteria selected fromEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspecies. In one embodiment, the gram-positive bacterial infection is aStaphylococcus infection. For example, the Staphylococcus infection isan S. aureus infection (e.g., a methicillin-resistant S. aureus (MRSA)infection). In one embodiment, the treatment regimen comprisesadministering an effective amount of a therapeutic, such as a compoundof formula (10), a pharmaceutically acceptable salt thereof, or ananalog thereof. In one embodiment, the dopamine receptor is selectedfrom DRD2, DRD2S, DRD2L, and DRD3. In one embodiment, the dopaminereceptor is from the D2-like family. In one embodiment, the dopaminereceptor is from the D1-like family. In one embodiment, the dopaminereceptor is DRD1. In one embodiment, the dopamine receptor is DRD2. Inone embodiment, the dopamine receptor is DRD3. In one embodiment, thedopamine receptor is DRD4. In one embodiment, the dopamine receptor isDRD5. In one embodiment, the dopamine receptor is DRD2, DRD3, or both.In one embodiment, the GPCR is a Class A GPCR. In one embodiment, theGPCR is GPR132. In one embodiment, the GPCR is selected from GPR132,GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, andcombinations thereof.

In one embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a compound of formula(10) or a pharmaceutically acceptable salt thereof, and an increasedDRD5 expression level measured in the sample relative to thepre-determined standard indicates that the treatment regimen is or isnot effective. In one embodiment, the dopamine receptor is DRD5, thetreatment regimen comprises administering an effective amount of atherapeutic, such as a compound of formula (10) or a pharmaceuticallyacceptable salt thereof, and mutation in the DRD5 gene measured in thesample indicates that the treatment regimen is or is not effective. Inone embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10) or a pharmaceutically acceptable salt thereof,and the misense mutation Q366R in the DRD5 gene measured in the sampleindicates that the treatment regimen is or is not effective.

In another aspect, provided herein are methods of identifying whether asubject having a condition is likely to be responsive to a treatmentregimen described herein. In one embodiment, the methods comprises (i)obtaining a biological sample from the subject; (ii) measuring gene copynumber or mutations in at least one dopamine receptor in the sample;(iii) comparing the copy number measured or mutations found in thesample to those for a pre-determined standard; and (iv) determiningwhether the subject is likely to be responsive to the treatment regimen,based on the copy number measured or mutations found in the sample tothose for the pre-determined standard. In one embodiment, the subjecthas, or is at risk of having, cancer. In one embodiment, the cancer is aneuro-oncology disease. In one embodiment, the cancer is aneuroendocrine tumor. In one embodiment, the cancer is selected from thegroup consisting of meningioma, ependymoma, glioma, neuroblastoma, anddiffuse intrinsic pontine glioma. In one embodiment, the subject has, oris at risk of having, a psychiatric disorder. For example, thepsychiatric disorder is selected from psychosis, schizophrenia, bipolardisorder, and major depressive disorder. In one embodiment, the subjecthas, or is at risk of having, an infection, such as a bacterialinfection. In one embodiment, the infection is a gram-negative bacterialinfection. In one embodiment, the infection is a gram-positive bacterialinfection. In one embodiment, the infection is an infection of abacteria selected from Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,and Enterobacter species. In one embodiment, the gram-positive bacterialinfection is a Staphylococcus infection. For example, the Staphylococcusinfection is an S. aureus infection (e.g., a methicillin-resistant S.aureus (MRSA) infection). In one embodiment, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10), a pharmaceutically acceptable salt thereof, oran analog thereof. In one embodiment, the dopamine receptor is from theD2-like family of dopamine receptors. In one embodiment, the dopaminereceptor is DRD1. In one embodiment, the dopamine receptor is DRD2. Inone embodiment, the dopamine receptor is DRD3. In one embodiment, thedopamine receptor is DRD4. In one embodiment, the dopamine receptor isDRD5. In one embodiment, the dopamine receptor is DRD2, DRD3, or both.In one embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10) or a pharmaceutically acceptable salt thereof,and mutation in the DRD5 gene measured in the sample indicates that thesubject is or is not likely to be responsive to the treatment regimen.In one embodiment, the dopamine receptor is DRD5, the treatment regimencomprises administering an effective amount of a therapeutic, such as acompound of formula (10) or a pharmaceutically acceptable salt thereof,and the misense mutation Q366R in the DRD5 gene measured in the sampleindicates that the subject is or is not likely to be responsive to thetreatment regimen.

In addition, measuring expression, post-translational modifications, oractivity levels of or mutations in eIF2-α, ATF4, CHOP, DR5, or cleavedor total cytokeratin 18 may be used to predict response or sensitivityto a method of treatment described herein and to identify subjectslikely to be responsive to a method of treatment described herein, suchas treatment with a compound of formula (10), a pharmaceuticallyacceptable salt thereof, or an analog thereof. In addition, measuringexpression, post-translational modifications, or activity levels of ormutations in eIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18can be used to assess the effectiveness of or monitor a method oftreatment described herein. Furthermore, measuring expression,post-translational modifications, or activity levels of or mutations ineIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be usedto screen in vivo, in vitro, or in silico for structurally unrelatedanti-cancer molecules. For example, competition and other assays knownin the art may be used to identify drugs able to outcompete the targetinteraction with a higher affinity to compare changes in those levels tothe respective changes produced by a compound of formula (10) or ananalog thereof. Assays can also be performed on living mammalian cells,which more closely approximate the effects of a particular serum druglevel in the body, or on microsomal extracts prepared from cultured celllines.

In one embodiment, the subject has, or is at risk of having, cancer. Inone embodiment, the treatment regimen comprises administering aneffective amount of an imipridone, such as ONC201, or an analog thereof.In one embodiment, the treatment regimen comprises administering aneffective amount of ONC201. In one embodiment, the treatment regimencomprises administering an effective amount of a compound of formula(10). In one embodiment, the compound of formula (10) is a compound offormula (40), e.g., a compound of formula (45). In one embodiment, acompound of formula (10) is a compound of formula (50), e.g., a compoundformula (55). In one embodiment, the compound of formula (10) is acompound of formula (80). In one embodiment, the compound of formula(10) is a compound of formula (90). In one embodiment, the compound offormula (10) is a compound of formula (60). In one embodiment, analogsof compound (1) have a structure selected from the structures of formula(25), formula (26), formula (27), formula (28), formula (29), formula(30), or formula (31).

Levels for a pre-determined standard can be, e.g., the average or medianlevels measured in samples from subjects. The levels for apre-determined standard can be measured under the same or substantiallysimilar experimental conditions as in measuring a sample from a subject.The levels for the pre-determined standard may be obtained from subjectswho are responsive to treatment with an imipridone, such as ONC201, oran analog thereof. In one embodiment, the pre-determined standard isobtained from subjects who are responsive to treatment with thecompound, and if the levels in a sample from a subject and in thestandard are similar, then the subject can be classified as likely to beresponsive to treatment. The levels for the pre-determined standard maybe obtained from subjects who are not responsive to treatment with thecompound. In one embodiment, the pre-determined standard is obtainedfrom subjects who are not responsive to treatment with the compound, andif the levels in a sample from a subject and in the pre-determinedstandard are different (e.g., up- or down-regulated), then the subjectcan be classified as likely to be responsive to treatment. The levelsfor the pre-determined standard may be obtained from normal healthysubjects.

Immunoassays can be used to assay protein or methylation levels in asample, including enzyme-linked immunosorbent assay (ELISA),enzyme-linked immunofiltration assay (ELIFA), flow cytometry,immunoblot, immunoprecipitation, immunohistochemistry,immunocytochemistry, luminescent immunoassay (LIA), fluorescentimmunoassay (FIA), and radioimmunoassay. m⁶A mRNA methylation levels canbe obtained by methylated RNA immunoprecipitation (Me-RIP)) or otherquantitative biochemical assays known in the art.

Nucleic acid mutations can be determined by any of a number ofprocedures. For example, a biologic sample from a subject is beobtained. Non-limiting examples of biological samples include a bodilyfluid (e.g., urine, saliva, plasma, or serum) or tissue sample (e.g., abuccal tissue sample or buccal cell). The biologic sample can then besequenced or scanned using known methods. For example, DNA arrays can beused to analyze at least a portion of the subject's genomic sequence.Furthermore, whole or partial genome sequence information can be used.Such sequences can be determined using standard sequencing methodsincluding chain-termination (Sanger dideoxynucleotide), dye-terminatorsequencing, and SOLID™ sequencing (Applied Biosystems). Whole genomesequences can be cut by restriction enzymes or sheared (mechanically)into shorter fragments for sequencing. DNA sequences can also beamplified using methods such as PCR and vector-based cloning methods(e.g., Escherichia coli). In one embodiment, at least a portion of asubject's genetic material (e.g., DNA, RNA, mRNA, cDNA, other nucleotidebases or derivatives of these) is scanned or sequenced using, e.g.,conventional DNA sequencers or chip-based technologies, to identify thepresence or absence of mutations or copy number variations.

In one aspect, provided herein are methods of identifying and treating asubject having a condition and who is likely to be responsive to atreatment regimen described herein. In one embodiment, the methodcomprises (i) identifying whether a subject having a condition is likelyto be responsive to a treatment regimen described herein; and (ii)treating with the treatment regimen a subject determined likely to beresponsive to that treatment regimen. In one embodiment, the subjecthas, or is at risk of having, cancer. In one embodiment, the treatmentregimen comprises administering an effective amount an imipridone, e.g.,ONC201, or an analog thereof. In one embodiment, the treatment regimencomprises administering an effective amount of compound (1). In oneembodiment, the treatment regimen comprises administering an effectiveamount of a compound of formula (10). In one embodiment, the compound offormula (10) is a compound of formula (40), e.g., a compound of formula(45). In one embodiment, a compound of formula (10) is a compound offormula (50), e.g., a compound formula (55). In one embodiment, thecompound of formula (10) is a compound of formula (80). In oneembodiment, the compound of formula (10) is a compound of formula (90).In one embodiment, the compound of formula (10) is a compound of formula(60). In one embodiment, analogs of compound (1) have a structureselected from the structures of formula (25), formula (26), formula(27), formula (28), formula (29), formula (30), or formula (31).

Levels for a pre-determined standard can be, e.g., the average or medianlevels measured in samples from subjects. The levels for apre-determined standard can be measured under the same or substantiallysimilar experimental conditions as in measuring a sample from a subject.The levels for the pre-determined standard may be obtained from subjectswho are responsive to treatment with an imipridone, such as ONC201, oran analog thereof. In one embodiment, the pre-determined standard isobtained from subjects who are responsive to treatment with thecompound, and if the levels in a sample from a subject are similar tothose in the standard, then the subject can be classified as likely tobe responsive to treatment. The levels for the pre-determined standardmay be obtained from subjects who are not responsive to treatment withthe compound. In one embodiment, the pre-determined standard is obtainedfrom subjects who are not responsive to treatment with the compound, andif the levels in a sample from a subject are different (e.g., up- ordown-regulated) than those in the pre-determined standard, then thesubject can be classified as likely to be responsive to treatment. Thelevels for the pre-determined standard may be obtained from normalhealthy subjects. Immunoassays can be used to assay protein levels in asample.

In one aspect, provided herein are methods of treating and assessing theeffectiveness of a treatment in a subject having a condition. In oneembodiment, the method comprises (i) treating the subject according to amethod of treatment described herein (ii) assessing as described hereinthe effectiveness of the treatment. In one embodiment, the subject has,or is at risk of having, cancer. In one embodiment, the treatmentregimen comprises administering an effective amount of an imipridone,such as ONC201, or an analog thereof. In one embodiment, the treatmentregimen comprises administering an effective amount of compound (1). Inone embodiment, the treatment regimen comprises administering aneffective amount of a compound of formula (10). In one embodiment, thecompound of formula (10) is a compound of formula (40), e.g., a compoundof formula (45). In one embodiment, a compound of formula (10) is acompound of formula (50), e.g., a compound formula (55). In oneembodiment, the compound of formula (10) is a compound of formula (80).In one embodiment, the compound of formula (10) is a compound of formula(90). In one embodiment, the compound of formula (10) is a compound offormula (60). In one embodiment, analogs of compound (1) have astructure selected from the structures of formula (25), formula (26),formula (27), formula (28), formula (29), formula (30), or formula (31).

Other conditions that may be suitable for the methods described hereininclude Attention Deficit Disorder; Addiction; Epilepsy; Viralinfection; Inflammation; Neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, Huntington's disease, Amyotrophic lateralsclerosis; Cardiovascular diseases such as coronary artery disease,cardiomyopathy, hypertensive heart disease, heart failure, pulmonaryheart disease, cardiac dysrhythmias, inflammatory heart disease,endocarditis, inflammatory cardiomegaly, myocarditis, valvular heartdisease, cerebrovascular disease, peripheral arterial disease,congenital heart disease, rheumatic heart disease; Diabetes; and lightchain amyloidosis.

V. Compositions

In one aspect, pharmaceutical compositions are provided, comprisingcompounds of formula (10):

or of formula (1):

and their pharmaceutically acceptable saltsIn one embodiment, the saltis a pharmaceutically acceptable mono-salt of the compound. In oneembodiment, the salt is a pharmaceutically acceptable di-salt of thecompound. In one embodiment, the salt is a pharmaceutically acceptablemono- or multi-salt (e.g., a di-salt or tri-salt) thereof selected fromhydrochloride, hydrobromide, hydrogensulphate, sulfates, phosphates,fumarates, succinates, oxalates and lactates, bisulfates, hydroxyl,tartrate, nitrate, citrate, bitartrate, carbonate, malate, maleate,fumarate sulfonate, methylsulfonate, formate, acetate, and carboxylate.In one embodiment, the salt is a salt selected from the group consistingof p-toluene-sulfonate, benzenesulfonate, citrate, methanesulfonate,oxalate, succinate, tartrate, fumarate and maleate. In one embodiment,the salt is a salt selected from the group consisting of ammonium,sodium, potassium, calcium, magnesium, zinc, lithium, and/or withcounter-ions such as methylamino, dimethylamino, diethylamino andtriethylamino counter-ions. In one embodiment, the salt is a.di-hydrochloride salt or a di-hydrobromide salt.

Compound (1) (ONC201) has the same chemical structure that would berevealed by structural analysis (e.g., NMR, X-ray diffraction) ofcompound NSC 350625, available from the National Cancer Institute'sDevelopmental Therapeutics Program Repository.

In one embodiment, the pharmaceutical composition includes a di-salt(e.g., a di-hydrochloride salt) of ONC201 or an analog thereof (e.g., animipridone). Salts (e.g., di-salts or tri-salts) of an ONC201 analog canbe prepared from an ONC201 analog, which can be synthesized as describedherein, or using standard chemical synthetic methodology known to one ofordinary skill in the art.

In one embodiment, the pharmaceutical composition includes at least onepharmaceutically acceptable carrier. Non-limiting examples of suitablepharmaceutically acceptable carriers include, those in Handbook ofPharmaceutical Excipients, 7^(th) ed., edited by Raymond C. Rowe et al.,American Pharmaceutical Association, Washington, USA and PharmaceuticalPress, London; and earlier editions. Exemplary pharmaceuticallyacceptable carriers, methods for making pharmaceutical compositions andvarious dosage forms, as well as administration modes are well-known inthe art, for example as detailed in Pharmaceutical Dosage Forms:Tablets, edited by Larry L. Augsburger & Stephen W. Hoag., London:Informa Healthcare, 2008; and in L. V. Allen, Jr. et al., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, 8^(th) ed.,Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004; A. R. Gennaro,Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins, 21^(st) ed., 2005, particularly chapter 89; and J. Hardman etal., Goodman & Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill Professional, 10^(th) ed., 2001.

In one embodiment, pharmacuetical compositions are formulated for ocularadministration. In one embodiment, pharmaceutical compositions areformulated for topical administration. In one embodiment, pharmaceuticalcompositions are formulated as drops, ointments, or liquids. In oneembodiment, pharmaceutical compositions include conventionalpharmaceutical carriers such as aqueous, powdery or oily bases,thickeners.

In one embodiment, a pharmaceutical composition is a formulation forintravenous administration. In one embodiment, the intravenousformulation comprises a compound of formula (10) or a pharmaceuticallyacceptable salt thereof dissolved in a solvent. In one embodiment, thesolvent comprises water. In one embodiment, the intravenous formulationincludes the compound or its salt in a concentration of about 0.05,about 0.25, about 0.5, about 2.5, about 5, about 25, or about 50 mg/mL.In one embodiment, the intravenous formulation includes the compound orits salt in a concentration of from about 0.05, 0.5, or 5 mg/mL to about1, 10, or 100 mg/mL. In one embodiment, the intravenous formulationincludes from about 0.005% 0.05%, or 0.5% to about 0.1%, 1%, or 10% ofthe compound or its salt. In one embodiment, the intravenous formulationincludes about 0.05%, 0.5%, or 5% of the compound or its salt. In oneembodiment, the intravenous formulation includes a higher or a lowerconcentration of the compound or its salt.

In one embodiment, the intravenous formulation has a pH of about 3. Inone embodiment, the formulation is adjusted to pH 3 with a phosphatebuffer. In one embodiment, the intravenous formulation includes dextroseor sodium chloride. In one embodiment, the intravenous formulationincludes the compound or its salt in a concentration of about 5 mg/mLand pH 3 and forms a stable solution. In one embodiment, the intravenousformulation includes the compound or its salt in a concentration ofabout 5 mg/mL and pH<5 and forms a stable solution. In one embodiment,the intravenous formulation includes the compound or its salt and one ormore antioxidants. In one embodiment, the intravenous formulationincludes a mixture of mono- and di-hydrochloride salts of the compound.In one embodiment, the intravenous formulation includes the compound orits salt as a 1% solution in a concentration of about 10 mg/mL. Forexample, the intravenous formulation is a solution with a pH of about3.3. In one embodiment, the pH is less than 4.0.

In one embodiment, a suitable pharmaceutically acceptable carrierincludes an aqueous carrier. In one embodiment, the aqueous carrierincludes sterile water. In one embodiment, the formulation includesdextrose, sodium chloride or both. In one embodiment, thepharmaceutically acceptable carrier includes an oil.

In one embodiment, an intravenous formulation comprises ONC201 or ananalog thereof or a di-hydrochloride salt thereof dissolved in water at25 mg/mL. In one embodiment, the formulation is adjusted to pH 3 withphosphate buffer. In one embodiment, the formulation includes dextrose,sodium chloride or both. In one embodiment, the formulation includes ahigher or a lower concentration of the di-hydrochloride salt of ONC201or an analog thereof. In one embodiment, the formulation includes ONC201or an analog thereof or a di-hydrochloride salt thereof in aconcentration of about 5 mg/mL. In one embodiment, the formulation ofabout 5 mg/mL forms a stable solution and pH 3. In one embodiment, theformulation of about 5 mg/mL has a pH<5 and forms a stable solution. Inone embodiment, the intravenous formulation includes ONC201 or an analogthereof or a di-hydrochloride salt thereof and one or more antioxidants.In one embodiment, the intravenous formulation includes a mixture ofmono- and di-hydrochloride salts of ONC201 or an analog thereof. In oneembodiment, the intravenous formulation includes ONC201 or an analogthereof or a di-hydrochloride salt thereof as a 1% solution in aconcentration of about 10 mg/mL. For example, the intravenousformulation is a solution having a pH of about 3.3. In one embodiment,the pH is less than 4.0.

In one embodiment, the intravenous formulation includes from about 0.5%to about 10% (or from about 5 mg/mL to about 100 mg/mL) of ONC201 or ananalog thereof or a di-salt thereof. In one embodiment, the formulationincludes from about 5% (or about 50 mg/mL) of ONC201 or an analogthereof or a di-salt thereof. In one embodiment, the intravenousinfusion rate may be slowed to decrease side effects of ONC201 or ananalog thereof or a di-salt thereof.

In one embodiment, the pharmaceutical composition comprises about0.1-99% of an ONC201 salt or an analog thereof; and a pharmaceuticallyacceptable carrier, e.g., an oil or sterile water or other aqueouscarrier. In one embodiment, the composition comprises a mono or di-saltof ONC201 or an analog thereof in a range of from about 5% to about 50%for oral dosage forms.

In one embodiment, a pharmaceutical composition includes an antioxidant.Suitable antioxidants include: ascorbic acid derivatives such asascorbic acid, erythorbic acid, sodium ascorbate, thiol derivatives suchas thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol,dithiothreitol, glutathione, tocopherols, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), sulfurous acid salts such assodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodiumformaldehyde sulfoxylate, sodium metabisulfite, sodium sulfite, andsodium thiosulfate, nordihydroguaiaretic acid. It should be noted thatantioxidants used for aqueous formulations typically include: sodiumsulphite, sodium metabisulphite, sodium formaldehyde sulphoxylate orascorbic acid and combinations thereof, whereas antioxidants used inoil-based solutions, organic solvents, include BHT, BHA or propylgallate and combinations thereof. In yet other embodiments, anantioxidant can be one or more of a flavanoid, an isoflavone,monothioglycerol, L-cysteine, thioglycolic acid, α-tocopherol, ascorbicacid 6-palmitate, dihydrolipoic acid, BHT, BHA, vitamin E, propylgallate, β-carotene, and ascorbic acid. Antioxidants can typically beused in about 0.1% to 1.0% by weight, more typically about 0.2%.

In one embodiment, the pharmaceutical composition includes animipridone, such as ONC201 or an analog thereof, or a pharmaceuticallyacceptable salt thereof and at least one other therapeutic agent. Forexample, the other therapeutic agent is selected from the groupconsisting of hormone analogs and antihormones, aromatase inhibitors,LHRH agonists and antagonists, inhibitors of growth factors, growthfactor antibodies, growth factor receptor antibodies, tyrosine kinaseinhibitors; antimetabolites; antitumour antibiotics; platinumderivatives; alkylation agents; antimitotic agents; tubuline inhibitors;PARP inhibitors, topoisomerase inhibitors, serine/threonine kinaseinhibitors, tyrosine kinase inhibitors, protein protein interactioninhibitors, RAF inhibitors, MEK inhibitors, ERK inhibitors, IGF-1Rinhibitors, ErbB receptor inhibitors, rapamycin analogs, BTK inhibitors,CRM1 inhibitors (e.g., KPT185), P53 modulators (e.g., Nutlins),antiangiogenics (e.g., axitinib, aflibercept, sorafenib, andregorafenib), amifostin, anagrelid, clodronat, filgrastin, interferon,interferon α, leucovorin, rituximab, procarbazine, levamisole, mesna,mitotane, pamidronate and porfimer, 2-chlorodesoxyadenosine,2-fluorodesoxy-cytidine, 2-methoxyoestradiol, 2C4,3-alethine,131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B,A 105972, A 204197, abiraterone, aldesleukin, alitretinoin,allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole,AG-2037, AP-5280, apaziquone, apomine, aranose, arglabin, arzoxifene,atamestane, atrasentan, auristatin PE, ABT-199 (Venetoclax), ABT-263(Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162,ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (selumetinib),ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055,AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib), avastin,AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate,BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797,BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992(afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI 6727,BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid, bleomycin A,bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin,busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell, calcitriol, canertinib,canfosfamide, capecitabine, carboxyphthalatoplatin, CC₁₋₇₇9, CC-115,CC-223, CEP-701, CEP-751, CBT-1 cefixime, ceflatonin, ceftriaxone,celecoxib, celmoleukin, cemadotin, CH4987655/RO-4987655,chlorotrianisene, cilengitide, ciclosporin, CDA-II, CDC-394, CKD-602,CKI-27, clofarabin, colchicin, combretastatin A4, COT inhibitors,CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin 52, CTP-37, CTLA-4monoclonal antibodies, CP-461, CV-247, cyanomorpholinodoxorubicin,cytarabine, D 24851, decitabine, deoxorubicin, deoxyrubicin,deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone,dexrazoxanet, diethylstilbestrol, diflomotecan, didox, DMDC, dolastatin10, doranidazole, DS-7423, E7010, E-6201, edatrexat, edotreotide,efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509,enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab,ER-86526, erlotinib, ET-18-0CH3, ethynylcytidine, ethynyloestradiol,exatecan, exatecan mesylate, exemestane, exisulind, fenretinide,figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI,formestane, fotemustine, galarubicin, gallium maltolate, gefinitib,gemtuzumab, gimatecan, glufosfamide, GCS-100, GDC-0623, GDC-0941(pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DTimmunogen, GMK, GPX-100, gp100-peptide vaccines, GSK-5126766,GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib),GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795,GW2016, granisetron, herceptine, hexamethylmelamine, histamine,homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesteronecaproate, ibandronate, ibrutinib, ibritumomab, idatrexate, idenestrol,IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), immunol,indisulam, interferon α-2a, interferon α-2b, pegylated interferon α-2b,interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab,iproplatin, irofulven, isohomohalichondrin-B, isoflavone, isotretinoin,ixabepilone, JRX-2, JSF-154, J-107088, conjugated oestrogens, kahalid F,ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim,leuprolide, leuporelin, lexidronam, LGD-1550, linezolid, lutetiumtexaphyrin, lometrexol, losoxantrone, LU 223651, lurtotecan, LY-S6AKT1,LY-2780301, mafosfamide, marimastat, mechloroethamine, MEK inhibitors,MEK-162, methyltestosteron, methylprednisolone, MEDI-573, MEN-10755,MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid,mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,regorafenib, revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440,rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab,rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone,rubitecan, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA,sargramostim, satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668,SDX-101, semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897,SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment, the other therapeutic agent comprises a hormoneanalog, an antihormone or both selected from tamoxifen, toremifene,raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide,bicalutamide, aminoglutethimide, cyproterone acetate, finasteride,buserelin acetate, fludrocortisone, fluoxymesterone,medroxy-progesterone, octreotide, and combinations thereof. In oneembodiment, the other therapeutic agent comprises one or more LHRHagonists are selected from goserelin acetate, luprolide acetate,triptorelin pamoate and combinations thereof and wherein the LHRHantagonists are selected from Degarelix, Cetrorelix, Abarelix, Ozarelix,Degarelix combinations thereof. In one embodiment, the other therapeuticagent comprises one or more growth factor inhibitors selected frominhibitors of: platelet derived growth factor (PDGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), epidermalgrowth factor (EGF), insuline-like growth factors (IGF), human epidermalgrowth factor (HER) and hepatocyte growth factor (HGF). In oneembodiment, the other therapeutic agent comprises one or more inhibitorsof the human epidermal growth factor selected from HER2, HER3, and HER4.In one embodiment, the other therapeutic agent comprises one or moretyrosine kinase inhibitors selected from cetuximab, gefitinib, imatinib,lapatinib and trastuzumab, and combinations thereof. In one embodiment,the other therapeutic agent comprises one or more aromatase inhibitorsselected from anastrozole, letrozole, liarozole, vorozole, exemestane,atamestane, and combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more antimetabolites which areantifolates selected from methotrexate, raltitrexed, and pyrimidineanalogs. In one embodiment, the other therapeutic agent comprises one ormore antimetabolites which are pyrimidine analogs selected from5-fluorouracil, capecitabin and gemcitabin. In one embodiment, the othertherapeutic agent comprises one or more antimetabolites which are purineand/or adenosine analogs selected from mercaptopurine, thioguanine,cladribine and pentostatin, cytarabine, fludarabine, and combinationsthereof. In one embodiment, the other therapeutic agent comprises one ormore antitumour antibiotics selected from anthracyclins, doxorubicin,daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin,dactinomycin, plicamycin, streptozocin and combinations thereof. In oneembodiment, the other therapeutic agent comprises one or more platinumderivatives selected from cisplatin, oxaliplatin, carboplatin andcombinations thereof. In one embodiment, the other therapeutic agentcomprises one or more alkylation agents selected from estramustin,meclorethamine, melphalan, chlorambucil, busulphan, dacarbazin,cyclophosphamide, ifosfamide, temozolomide, nitrosoureas, andcombinations thereof. In one embodiment, the other therapeutic agentcomprises nitrosoureas selected from carmustin, lomustin, thiotepa, andcombinations thereof. In one embodiment, the other therapeutic agentcomprises antimitotic agents selected from Vinca alkaloids and taxanes.In one embodiment, the other therapeutic agent comprises one or moretaxanes selected from paclitaxel, docetaxel, and combinations thereof.In one embodiment, the other therapeutic agent comprises one or moreVinca alkaloids selected from vinblastine, vindesin, vinorelbin,vincristine, and combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more topoisomerase inhibitors whichare epipodophyllotoxins. In one embodiment, the other therapeutic agentcomprises one or more epipodophyllotoxins selected from etoposide andetopophos, teniposide, amsacrin, topotecan, irinotecan, mitoxantron, andcombinations thereof. In one embodiment, the other therapeutic agentcomprises one or more serine/threonine kinase inhibitors selected fromPDK 1 inhibitors, B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors,PI3K inhibitors, dual mTOR/PI3K inhibitors, STK 33 inhibitors, AKTinhibitors, PLK 1 inhibitors, inhibitors of CDKs, Aurora kinaseinhibitors, and combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more tyrosine kinase inhibitors whichare PTK2/FAK inhibitors. In one embodiment, the other therapeutic agentcomprises one or more protein protein interaction inhibitors selectedfrom IAP, Mcl-1, MDM2/MDMX and combinations thereof. In one embodiment,the other therapeutic agent comprises one or more rapamycin analogsselected from everolimus, temsirolimus, ridaforolimus, sirolimus, andcombinations thereof. In one embodiment, the other therapeutic agentcomprises one or more therapeutic agents selected from amifostin,anagrelid, clodronat, filgrastin, interferon, interferon α, leucovorin,rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate andporfimer, and combinations thereof. In one embodiment, the othertherapeutic agent comprises one or more therapeutic agents selected from2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyoestradiol,2C4,3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin,16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin,alitretinoin, allovectin-7, altretamine, alvocidib, amonafide,anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, aranose,arglabin, arzoxifene, atamestane, atrasentan, auristatin PE, ABT-199(Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479(ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244(selumetinib), ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717,AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib),avastin, AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006,BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodardicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550,BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid,bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib,brostallicin, busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell,calcitriol, canertinib, canfosfamide, capecitabine,carboxyphthalatoplatin, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1cefixime, ceflatonin, ceftriaxone, celecoxib, celmoleukin, cemadotin,CH4987655/RO-4987655, chlorotrianisene, cilengitide, ciclosporin,CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicin, combretastatinA4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin52, CTP-37, CTLA-4 monoclonal antibodies, CP-461, CV-247,cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine,deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide,desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol,diflomotecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010,E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFRinhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin,epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3,ethynylcytidine, ethynyloestradiol, exatecan, exatecan mesylate,exemestane, exisulind, fenretinide, figitumumab, floxuridine, folicacid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin,gallium maltolate, gefinitib, gemtuzumab, gimatecan, glufosfamide,GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068,GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100-peptidevaccines, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436(dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183,GSK-2141795, GW2016, granisetron, herceptine, hexamethylmelamine,histamine, homoharringtonine, hyaluronic acid, hydroxyurea,hydroxyprogesterone caproate, ibandronate, ibrutinib, ibritumomab,idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12(cixutumumab), immunol, indisulam, interferon α-2a, interferon α-2b,pegylated interferon α-2b, interleukin-2, INK-1117, INK-128, INSM-18,ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B,isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088,conjugated oestrogens, kahalid F, ketoconazole, KW-2170, KW-2450,lobaplatin, leflunomide, lenograstim, leuprolide, leuporelin,lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol,losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide,marimastat, mechloroethamine, MEK inhibitors, MEK-162,methyltestosteron, methylprednisolone, MEDI-573, MEN-10755, MDX-H210,MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin,mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb,rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453,RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan,R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim,satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101,semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747,SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment, the other therapeutic agent comprises a steroid,including dexamethasone, prednisolone, methyl prednisolone, prednisone,hydrocortisone, triamcinolone, betamethasone, and cortivazol. In oneembodiment, the other therapeutic agent comprises an anti-emetic,Anti-emetics include, but are not limited to, 5-HT3 receptor agonists(e.g., dolasetron, granisetron, ondansetron, tropisetron, palonosetron,and mirtazapine), dopamine agonists (e.g., domperidone, olanzapine,droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride,prochlorperazine, and metoclopramide), NK1 receptor antagonists (e.g.,aprepitant and casopitant), antihistamines (such as cyclizine,diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine,hydroxyzine), cannabinoids (e.g., cannabis, dronabinol, nabilone, andsativex), benzodiazepines (e.g., midazolam and lorazepam),anticholinergics (e.g., hyoscine), trimethobenzamide, ginger, emetrol,propofol, peppermint, muscimol, and ajwain.

In one embodiment, the other therapeutic agent comprises an anti-canceragent, which includes a mitotic inhibitor. In one embodiment, themitotic inhibitor includes a taxane. In one embodiment, the mitoticinhibitor includes a taxane selected from paclitaxel and docetaxel.

In one embodiment, the pharmaceutical composition includes animipridone, such as ONC201, or an analog thereof, or a pharmaceuticallyacceptable salt thereof; and at least one anti-cancer agent, whichincludes one or more of acivicin, aclarubicin, acodazole, acronine,adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine,ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine,anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin,azacitidine, azetepa, azotomycin, batimastat, benzodepa, bevacizumab,bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin,brequinar, bropirimine, busulfan, cactinomycin, calusterone,capecitabine, caracemide, carbetimer, carboplatin, carmustine,carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin,dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin,droloxifene, dromostanolone, duazomycin, edatrexate, eflomithine,elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin,erbulozole, esorubicin, estramustine, etanidazole, etoposide, etoprine,fadrozole, fazarabine, fenretinide, floxuridine, fludarabine,fluorouracil, flurocitabine, fosquidone, fostriecin, fulvestrant,gemcitabine, hydroxyurea, idarubicin, ifosfamide, ilmofosine,interleukin II (IL-2, including recombinant interleukin II or rIL2),interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3,interferon β-Ia, interferon gamma-Ib, iproplatin, irinotecan,lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine,losoxantrone, masoprocol, maytansine, mechlorethamine hydrochlride,megestrol, melengestrol acetate, melphalan, menogaril, mercaptopurine,methotrexate, metoprine, meturedepa, mitindomide, mitocarcin,mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane,mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogalamycin,ormnaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin,pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan,piroxantrone hydrochloride, plicamycin, plomestane, porfimer,porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin,riboprine, rogletimide, safingol, semustine, simtrazene, sparfosate,sparsomycin, spirogermanium, spiromustine, spiroplatin, streptonigrin,streptozocin, sulofenur, talisomycin, tamoxifen, tecogalan, tegafur,teloxantrone, temoporfin, teniposide, teroxirone, testolactone,thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan,toremifene, trestolone, triciribine, trimetrexate, triptorelin,tubulozole, uracil mustard, uredepa, vapreotide, verteporfin,vinblastine, vincristine sulfate, vindesine, vinepidine, vinglycinate,vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole,zeniplatin, zinostatin, zoledronate, zorubicin and combinations thereof.

Examples of suitable anti-cancer agents include those described Goodmanand Gilman's The Pharmacological Basis of Therapeutics, 12^(th) Ed.,edited by Laurence Brunton, Bruce Chabner, Bjorn Knollman, McGraw HillProfessional, 2010.

In some exemplary embodiments, the pharmaceutical composition includes asalt (e.g., a mono- or di-salt) of an imipridone, e.g., ONC201, or ananalog thereof and at least one other therapeutic agent, where the othertherapeutic agent comprises an anti-angiogenic agent, for example,bevacizumab. In one embodiment, the anti-angiogenic agent is selectedfrom aflibercept, axitinib, angiostatin, endostatin, 16 kDa prolactinfragment, laminin peptides, fibronectin peptides, tissuemetalloproteinase inhibitors (TIMP 1, 2, 3, 4), plasminogen activatorinhibitors (PAI-1, -2), tumor necrosis factor α, (high dose, invitro),TGF-β1, interferons (IFN-α, -β, γ), ELR-CXC chemokines, IL-12; SDF-1;MIG; platelet factor 4 (PF-4); IP-10, thrombospondin (TSP), SPARC,2-methoxyoestradiol, proliferin-related protein, suramin, sorafenib,regorafenib, thalidomide, cortisone, linomide, fumagillin (AGM-1470;TNP-470), tamoxifen, retinoids, CM101, dexamethasone, leukemiainhibitory factor (LIF), hedgehog inhibitor and combinations thereof.

A pharmaceutical combination can include first and second therapeuticagents in any desired proportions provided that the synergistic orcooperative effect still occurs. A synergistic pharmaceuticalcombination preferably contains the first and second therapeutic agentsin a ratio of from about 1:9 to about 9:1. In one embodiment, asynergistic combination contains the first and second therapeutic agentsin a ratio of from about 1:8 to about 8:1, from about 1:7 to about 7:1,from about 1:6 to about 6:1, from about 1:5 to about 5:1, from about 1:4to about 4:1, from about 1:3 to about 3:1, or from about 1:2 to about2:1. In one embodiment, the synergistic combination contains thetherapeutic agents in a ratio of approximately 1:1.

In one embodiment, the second therapeutic agent is selected fromAllopurinol, Arsenic Trioxide, Azacitidine, Bortezomib, Bevacizumab,Capecitabine, Carboplatin, Celecoxib, Chlorambucil, Clofarabine,Cytarabine, Dacarbazine, Daunorubicin HCl, Docetaxel, Doxorubicin HCl,Floxuridine, Gemcitabine HCl, Hydroxyurea, Ifosfamide, ImatinibMesylate, Ixabepilone, Lenalidomide, Megestrol acetate, Methotrexate,Mitotane, Mitoxantrone HCl, Oxaliplatin, Paclitaxel, Pralatrexate,Romidepsin, Sorafenib, Streptozocin, Tamoxifen Citrate, Topotecan HCl,Tretinoin, Vandetanib, Vismodegib, Vorinostat, and combinations thereof.

In one embodiment, the second therapeutic agent comprises a smallmolecule multi-kinase inhibitor, e.g., sorafenib or regorafenib. In oneembodiment, the second therapeutic agent comprises a Hedgehog PathwayInhibitor, e.g., vismodegib. In one embodiment, the second therapeuticagent includes a drug selected from Table 2 below.

TABLE 2 Classes Of Drugs Classes of drugs Examples Purine allopurinol,oxypurinol, clofarabine, and tisopurine analogs Pyrimidine5-fluorouracil, Floxuridine (FUDR), capecitabine, analogs cytarabine,6-azauracil (6-AU), and gemcitabine (Gemzar) Proteasome bortezomib,carfilzomib, cecliranib, disulfiram, inhibitorsepigallocatechin-3-gallate, salinosporamide A, ONCX 0912, CEP-18770,MLN9708, epoxomicin, and MG132. Anti- bevacizumab, aflibercept,sunitinib, sorafenib, pazopanib, angiogenic vandetanib, cabozantinib,axitinib, ponatinib, regorafenib, ranibizumab, lapatinib, andvandetanib. Platinum- cisplatin, carboplatin, oxaliplatin, satraplatin,picoplatin, based nedaplatin, and triplatin. antineo- plastic drugsCOX-2 celecoxib, valdecoxib (Bextra), parecoxib (Dynastat), inhibitorslumiracoxib, etoricoxib, and rofecoxib. Nitrogen cyclophosphamide,chlorambucil, uramustine, ifosfamide, mustards melphalan, bendamustine,and mustine. Alkylating cyclophosphamide, mechlorethamine or mustine(HN2) agents (trade name Mustardgen), uramustine or uracil mustard,melphalan, chlorambucil, ifosfamide, bendamustine, carmustine,lomustine, streptozocin, and busulfan. Anthra- Daunorubicin(Daunomycin), Daunorubicin (liposomal), cyclines Doxorubicin(Adriamycin), Doxorubicin (liposomal), Epirubicin, Idarubicin,Valrubicin, and Mitoxantrone. Taxanes Paclitaxel (Taxol), Docetaxel(Taxotere), and albumin-bound paclitaxel (Abraxane). Nucleotidemethotrexate, pralatrexate, hydroxyurea, and 5- synthesisfluorodeoxyuridine, 3,4-dihydroxybenzylamine Bcr-abl imatinib,nilotinib, dasatinib, bosutinib and ponatinib. inhibitors Other arsenictrioxide, thalidomide, revlimid, and mitotane. Topo- amsacrine,etoposide, etoposide phosphate, teniposide, isomerase doxorubicin,Topotecan (Hycamtin), Irinotecan (CPT-11, inhibitor Camptosar),Exatecan, Lurtotecan, ST 1481, CKD 602, ICRF-193, and genistein. HDACVorinostat (SAHA), Romidepsin (Istodax), Panobinostat inhibitors(LBH589), Valproic acid (as Mg valproate), Belinostat (PXD101),Mocetinostat (MGCD0103), Abexinostat (PCI- 24781), Entinostat (MS-275),SB939, Resminostat (4SC- 201), Givinostat, Quisinostat (JNJ-26481585),CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215,ME-344, sulforaphane, Kevetrin, and ATRA. Multi- sorafenib, regorafenib,and vandetanib. kinase inhibitors Hormone tamoxifen, toremifene,Arimidex (anastrozole), Aromasin therapies (exemestane), Femara(letrozole), and Fulvestrant (Faslodex). Hedgehog vismodegib,BMS-833923, IPI-926, LDE-225, PF-04449913, signaling LEQ 506, andTAK-441. Inhibitors Checkpoint Opdivo (nivolumab), Durvalumab(Medi4736), Keytruda Inhibitors (pembrolizumab, MK3475), BGB-A317,AMP-224, PDR001, REGN 281, Atezolizumab (MPDL3280A), Pidilizumab(BMS-936559, CT-011, ONO-4538), Avelumab (MSB0010718 C), Yervoy(ipilimumab), tremelimumab BCL2 AT-101, Bcl-2/xL inhibitor, InhibitorsNavitoclax (ABT-263), Venetoclax (ABT-199), Apogossypol, PTN1258,obatoclax, G3139

In one embodiment, the second therapeutic agent includes drugs thattarget tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)receptors. In one embodiment, the second therapeutic agent includesrecombinant TRAIL or an agonistic antibody that activates one or moreTRAIL receptors. In one embodiment, the second therapeutic agentincludes one or more antibodies or recombinant TRAIL that activatesignaling by DR4, DR5 or both. In one embodiment, the second therapeuticagent includes one or more of AMG-655, LBY-135, mapatumumab,lexatumumab, Apomab, and rhApo2L/TRAIL. In one embodiment, the secondtherapeutic agent includes an active agent selected from Camptothecin,5-FU, capecitabine, cisplatin, doxorubicin, irinotecan, paclitaxel,cisplatin, bortezomib, BH3I-2, rituximab, radiation, triterpenoids,sorafenib, gemcitabine, HDAC inhibitors, carboplatin, T-101 (a gossypolderivate), ABT-263, ABT-737, and GX-15-070 (obatoclax), vorinostat,cetuximab, panitumumab, bevacizumab, ganitumab, interferon gamma,sorafenib, XIAP antagonists, Bcl-2 antagonists, and Smac mimetics.

VI. Dose

In one embodiment, a pharmaceutical composition comprises an imipridone,such as ONC201, or an analog thereof, or a pharmaceutically acceptablesalt thereof in a dose ranging from about 40, 50, 60, or 100 mg to about2000 mg; from about 4, 5, 6, or 10 mg to about 200 mg; or from about0.4, 0.5, 0.6, or 1 mg to about 20 mg where the weight can be based onthe compound in its free base form. In one embodiment, a pharmaceuticalcomposition comprises an imipridone, such as ONC201, or an analogthereof, or a pharmaceutically acceptable salt thereof in a dose levelranging from about 50 mg to about 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000mg; from about 5 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, and 200 mg; or from about 0.5 mgto about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,and 20 mg. In one embodiment, a pharmaceutical composition comprises animipridone, such as ONC201, or an analog thereof, or a pharmaceuticallyacceptable salt thereof in a dose level ranging from about 40 mg toabout 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about 4 mg to about20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, or 200 mg; or from about 0.4 mg to about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 mg. In one embodiment,a pharmaceutical composition comprises an imipridone, such as ONC201, oran analog thereof, or a pharmaceutically acceptable salt thereof in adose level ranging from about 60 mg to about 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, or 2000 mg; from about 6 mg to about 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg; or fromabout 0.6 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 mg. In one embodiment, a pharmaceuticalcomposition comprises an imipridone, such as ONC201, or an analogthereof, or a pharmaceutically acceptable salt thereof in a dose levelranging from about 100 mg to about 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 mg, or2000 mg; from about 10 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg; or from about 1mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 mg. In one embodiment, a pharmaceutical composition comprisesan imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof in a dose level ranging fromabout 200 mg to about 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about20 mg to about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, or 200 mg; or from about 2 mg to about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg, based on thecompound in its free base form. In one embodiment, a pharmaceuticalcomposition comprises an imipridone, such as ONC201, or an analogthereof, or a pharmaceutically acceptable salt thereof in a dose levelranging from about 400 mg to about 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about40 mg to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200 mg; or from about 4 mg to about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg based on the compound inits free base form. In one embodiment, a pharmaceutical compositioncomprises an imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof thereof in a dose level rangingfrom about 50 mg to about 60, 70, 80, 90, or 100 mg; from about 60 mg toabout 70, 80, 90, or 100 mg; from about 70 mg to about 80, 90 or 100 mg,from about 80 mg to about 90 or 100 mg; from about 90 mg to about 100mg; from about 5 mg to about 6, 7, 8, 9, or 10 mg; from about 6 mg toabout 7, 8, 9, or 10 mg; from about 7 mg to about 8, 9 or 10 mg, fromabout 8 mg to about 9 or 10 mg; from about 9 mg to about 10 mg; fromabout 0.5 mg to about 0.6, 0.7, 0.8, 0.9, or 1 mg; from about 0.6 mg toabout 0.7, 0.8, 0.9, or 1 mg; from about 0.7 mg to about 0.8, 0.9 or 1mg, from about 0.8 mg to about 0.9 or 1 mg; or from about 0.9 mg toabout 1 mg.

In one embodiment, a pharmaceutical composition comprises an imipridone,such as ONC201, or an analog thereof, or a pharmaceutically acceptablesalt thereof in a dose ranging from about 1 to about 40 mg/kg; about 0.1to about 4 mg/kg; or about 0.01 to about 0.40 mg/kg. In one embodiment,a pharmaceutical composition comprises an imipridone, such as ONC201, oran analog thereof, or a pharmaceutically acceptable salt thereof in adose level ranging from about 1, 2, 3, 4, 5, 6, 7, 8, or 9 mg/kg toabout 10, 20, 30, or 40 mg/kg; from about 10, 11, 12, 13, 14, 15, 16,17, 18, or 19 mg/kg to about 20, 30, or 40 mg/kg; from about 20, 21, 22,23, 24, 25, 26, 27, 28, or 29 mg/kg to about 30 or 40 mg/kg; from about30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 mg/kg to about 40 mg/kg; fromabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg/kg to about 1,2, 3, or 4 mg/kg; from about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, or 1.9 mg/kg to about 2, 3, or 4 mg/kg; from about 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, or 2.9 mg/kg to about 3 or 4 mg/kg; orfrom about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, or 3.9 mg/kg toabout 4 mg/kg; from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09 mg/kg to about 0.10, 0.20, 0.30, or 0.40 mg/kg; from about0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, or 0.19 mg/kg toabout 0.20, 0.30, or 0.40 mg/kg; from about 0.20, 0.21, 0.22, 0.23,0.24, 0.25, 0.26, 0.27, 0.28, or 0.29 mg/kg to about 0.30 or 0.40 0.mg/kg; or from about 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37,0.38, or 0.39 mg/kg to about 0.40 mg/kg.

In one embodiment, a pharmaceutical composition comprises an imipridone,such as ONC201, or an analog thereof, or a pharmaceutically acceptablesalt thereof in a dose ranging from about 37.5 mg/m² to about 1500mg/m²; from about 3.75 mg/m² to about 150 mg/m²; or from about 0.4 mg/m²to about 15 mg/m² In one embodiment, a pharmaceutical compositioncomprises comprises an imipridone, such as ONC201, or an analog thereof,or a pharmaceutically acceptable salt thereof in a dose ranging fromabout 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320,325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390,395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460,465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530,535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600,605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740,745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810,815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880,885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950,955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015,1020, 1025, 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075,1080, 1085, 1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135,1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195,1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255,1260, 1265, 1270, 1275, 1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315,1320, 1325, 1330, 1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375,1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435,1440, 1445, 1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495mg/m² to about 1500 mg/m²; from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, or 149 mg/m² to about 150 mg/m²; or from about 0.5,1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 111, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 mg/m² to about15 mg/m².

VII. Dosage Forms

Pharmaceutical compositions for use in the methods described herein canbe formulated into a dosage form that can be administered to a patient.In some embodiments, the composition is in the form of an oral orparenteral dosage unit. In some embodiments, the composition is in theform of an oral dosage unit. In some embodiments, the oral dosage unitis fractionated into several, smaller doses, which are administered to asubject over a predetermined period of time in order to reduce toxicityof a therapeutic agent being administered. In some embodiments, an oraldosage unit is administered as a tablet or capsule comprising acontrolled release formulation that can include a plurality ofparticles, granules, pellets, minitablets or tablets. In someembodiments, the composition is in the form of a parenteral dosage unit.In some embodiments, the parenteral dosage unit is selected fromintravenous (IV), subcutaneous (SC), and intramuscular (M), rectal (PR)or transdermal dosage units. In some embodiments, the composition is ina dosage form selected from sterile solutions, suspensions,suppositories, tablets and capsules. In some embodiments, thecomposition is an oral dosage form selected from a tablet, caplet,capsule, lozenge, syrup, liquid, suspension and elixir. In someembodiments, the composition is in an oral dosage form selected fromtablets, hard shell capsules, soft gelatin capsules, beads, granules,aggregates, powders, gels, solids and semi-solids.

In some embodiments, suitable forms of pharmaceutical compositions foruse in the methods described herein include dermatological compositionsadapted for cutaneous topical administration. For example,dermatological compositions include a cosmetically or pharmaceuticallyacceptable medium. Dermatological compositions for topicaladministration can include ointments, lotions, creams, gels, drops,suppositories, sprays, liquids and powders. In some embodiments,conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners, skin enhancers can be necessary or desirable and thereforeused. Examples of suitable enhancers include ethers, such as diethyleneglycol monoethyl ether (available commercially as TRANSCUTOL®) anddiethylene glycol monomethyl ether; surfactants such as sodium laurate,sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkoniumchloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80), andlecithin; alcohols such as ethanol, propanol, octanol, benzyl alcohol;polyethylene glycol and esters thereof such as polyethylene glycolmonolaurate; amides and other nitrogenous compounds such as urea,dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone,1-methyl-2-pyrrolidone, ethanolamine, diethanolamine andtriethanolamine; terpenes; alkanones; and organic acids, particularlycitric acid and succinic acid. AZONE® and sulfoxides such as DMSO andCι0OMSO may also be used, but are less preferred.

In some embodiments, the composition is in a dosage form selected fromsustained release, controlled release, delayed release and responserelease forms.

VIII. Methods of Use

The compositions and methods described herein have utility in treatingmany disease conditions, including cancer (e.g., colorectal, brain, andglioblastoma). In one embodiment, the compositions and methods describedherein are used to treat diseases such as ocular melanoma, desmoplasticround cell tumor, chondrosarcoma, leptomengial disease, diffuse largeB-cell lymphoma, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, AIDS-Related Cancers, AIDS-Related Lymphoma,Anal or Rectal Cancer, Appendix Cancer, Astrocytomas, and AtypicalTeratoid/Rhabdoid Tumor. In one embodiment, the compositions and methodsdescribed herein are used to treat diseases such as Basal CellCarcinoma, Basal Cell Nevus Syndrome, Gorlin-Nevus Syndrome, Bile DuctCancer, Bladder Cancer, Bone Cancer, Osteosarcoma and Malignant FibrousHistiocytoma, Brain Tumor, Breast Cancer, Bronchial Tumors, BurkittLymphoma, and Spinal Cord Tumors. In one embodiment, the compositionsand methods described herein are used to treat diseases such asCarcinoid Tumor, Carcinoma of Unknown Primary, Central Nervous SystemAtypical Teratoid/Rhabdoid Tumor, Leptomeningeal Disease, CentralNervous System Embryonal Tumors, Central Nervous System Lymphoma,Cervical Cancer, Chordoma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Chronic Myeloproliferative Disorders, ColonCancer, Colorectal Cancer, Craniopharyngioma, and Cutaneous T-CellLymphoma (including Sezary syndrome and mycosis fungoides (MF)). In oneembodiment, the compositions and methods described herein are used totreat diseases such as Embryonal Tumors of Central Nervous System,Endometrial Cancer, Ependymoblastoma, Ependymoma, Esophageal Cancer,Ewing Sarcoma Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, and EyeCancer, including Intraocular Melanoma and Retinoblastoma. In oneembodiment, the compositions and methods described herein are used totreat diseases such as Gallbladder Cancer, Gastric (Stomach) Cancer,Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST),Germ Cell Tumor, Gestational Trophoblastic Tumor, and Glioma. In oneembodiment, the compositions and methods described herein are used totreat a cancer selected from the group consisting of Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer,Histiocytosis, Hodgkin Lymphoma, and Hypopharyngeal Cancer. In oneembodiment, the compositions and methods described herein are used totreat diseases such as Kaposi Sarcoma and Kidney (Renal Cell) Cancer. Inone embodiment, the compositions and methods described herein are usedto treat diseases such as Langerhans Cell Histiocytosis, LaryngealCancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, includingNon-Small Cell Lung Cancer, and Small Cell Lung Cancer, Non-HodgkinLymphoma, and Primary Central Nervous System Lymphoma. In oneembodiment, the compositions and methods described herein are used totreat diseases such as Waldenström's macroglobulinemia(lymphoplasmacytic lymphoma), Malignant Fibrous Histiocytoma of Bone andOsteosarcoma, Medulloblastoma, Medulloepithelioma, Melanoma, Merkel CellCarcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with OccultPrimary, Multiple Endocrine Neoplasia Syndrome, Mouth Cancer, MultipleMyeloma/Plasma Cell Neoplasm, Mycosis Fungoides, MyelodysplasticSyndromes, complex karyotype, blastic phase leukemia,Myelodysplastic/Myeloproliferative Neoplasms, Multiple Myeloma, andMyeloproliferative Disorders. In one embodiment, the compositions andmethods described herein are used to treat cancer. In one embodiment,the compositions and methods described herein are used to treat diseasessuch as Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,and Neuroblastoma. In one embodiment, the compositions and methodsdescribed herein are used to treat diseases such as Oral Cancer, Lip andOral Cavity Cancer, Oropharyngeal Cancer, Osteosarcoma and MalignantFibrous Histiocytoma of Bone, Ovarian Cancer, Ovarian Germ Cell Tumor,Ovarian Epithelial Cancer, and Ovarian Low Malignant Potential Tumor. Inone embodiment, the compositions and methods described herein are usedto treat diseases such as Pancreatic Cancer, Papillomatosis, ParanasalSinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer,Pharyngeal Cancer, Pineal Parenchymal Tumors of IntermediateDifferentiation, Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Pituitary Tumor, Pleuropulmonary Blastoma,Pregnancy and Breast Cancer, Primary Central Nervous System Lymphoma,and Prostate Cancer. In one embodiment, the compositions and methodsdescribed herein are used to treat a cancer selected from the groupconsisting of Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvisand Ureter, Respiratory Tract Carcinoma Involving the NUT Gene onChromosome 15, Retinoblastoma, and Rhabdomyosarcoma. In one embodiment,the compositions and methods described herein are used to treat highgrade prostate cancer. In one embodiment, the compositions and methodsdescribed herein are used to treat medium grade prostate cancer. In oneembodiment, the compositions and methods described herein are used totreat low grade prostate cancer. In one embodiment, the compositions andmethods described herein are used to treat castration-resistant prostatecancer. In one embodiment, the compositions and methods described hereinare used to treat a nervous system tumor. In one embodiment, thecompositions and methods described herein are used to treat a centralnervous system tumor. In one embodiment, the compositions and methodsdescribed herein are used to treat a peripheral nervous system tumor. Inone embodiment, the compositions and methods described herein are usedto treat a paraganglioma. In one embodiment, the compositions andmethods described herein are used to treat a pheochromocytoma.

In in vitro models, in animal models, and in human clinical trialscompound (1) (ONC201) has broad anti-cancer activity, low toxicityincluding few, if any, adverse effects, low genotoxicity, and highbioavailability including oral bioavailability. These features allow ONC201 and various analogs to be particularly well suited for pediatricpatients. These features also make ONC 201 and various analogsparticularly well suited for chronic therapy, for high risk patients,and to ensure long-lasting responses or stable disease or to preventdisease recurrence.

In another aspect, provided herein are methods of treating or preventingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount compound (1)

or a pharmaceutically acceptable salt thereof, wherein the cancer is amidline glioma having a histone H3 K27M mutation. In some embodiments,the cancer is selected from the group consisting of a diffuse intrinsicpontine glioma, a diffuse midline glioma, a spinal cord glioma, athalamic glioma, a brainstem glioma, and a cerebellar glioma. In someembodiments, the cancer is not a spinal cord tumor. In some embodiments,the histone H3 K27M mutation is H3.3 K27M or H3.1 K27M. In someembodiments, the histone H3 K27M mutation is in one or more histonegenes selected from H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C,HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J.In some embodiments, DRD2 is overexpressed in cancerous tissue. In someembodiments, DRD5 is underexpressed in cancerous tissue. In someembodiments, DRD2 is overexpressed and DRD5 is underexpressed incancerous tissue. In some embodiments, the subject is a human. In someembodiments, the subject is a domesticated pet. In some embodiments, thesubject is a pediatric subject.

In another aspect, provided herein are methods of treating or preventingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof,wherein the cancer has a histone H3 mutation. In some embodiments, thecancer is selected from the group consisting of a central nervous systemtumor, a brain tumor, a peripheral nervous system tumor, apheochromocytoma, a paraganglioma, an adrenal cortical carcinoma, anadrenal tumor, and a neuroendocrine tumor. In some embodiments, thecancer is selected from the group consisting of meningioma, ependymoma,glioma, neuroblastoma, and diffuse intrinsic pontine glioma. In someembodiments, the cancer is selected from the group consisting of adiffuse midline glioma, a spinal cord glioma, a thalamic glioma, abrainstem glioma, and a cerebellar glioma. In some embodiments, thehistone H3 mutation is H3.3 K27M or H3.1 K27. In some embodiments, thecancer has a K27M mutation in one or more histone genes selected fromH3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E,HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J. In someembodiments, DRD2 is overexpressed in cancerous tissue. In someembodiments, DRD5 is underexpressed in cancerous tissue. In someembodiments, DRD2 is overexpressed and DRD5 is underexpressed incancerous tissue. In some embodiments, the compound is ONC201. In someembodiments, the subject is a human. In some embodiments, the subject isa domesticated pet. In some embodiments, the subject is a pediatricsubject.

In another aspect, provided herein are methods of treating or preventingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof,wherein the cancer is a midline glioma. In some embodiments, the canceris selected from the group consisting of a diffuse intrinsic pontineglioma, a diffuse midline glioma, a spinal cord glioma, a thalamicglioma, a brainstem glioma, and a cerebellar glioma. In someembodiments, the cancer is not a spinal cord tumor. In some embodiments,the cancer has a histone H3 mutation, wherein the histone H3 mutation isH3.3 K27M or H3.1 K27M. In some embodiments, the cancer has a histone H3K27M mutation in one or more histone genes selected from H3F3A, H3F3B,HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G,HIST1H3H, HIST1H3I, or HIST1H3J. In some embodiments, DRD2 isoverexpressed in cancerous tissue. In some embodiments, DRD5 isunderexpressed in cancerous tissue. In some embodiments, DRD2 isoverexpressed and DRD5 is underexpressed in cancerous tissue. In someembodiments, the compound is ONC201. In some embodiments, the subject isa human. In some embodiments, the subject is a domesticated pet. In someembodiments, the subject is a pediatric subject.

In one embodiment, the compositions and methods described herein areused to treat a pediatric cancer (e.g., pediatric solid tumors,pediatric sarcomas, pediatric Ewing's sarcomas, pediatric gliomas,pediatric central nervous system cancers, pediatric neuroblastoma,pediatric leukemia and pediatric lymphoma).

In one embodiment, the compositions and methods described herein areused to treat a proliferative skin disorder such as psoriasis. In oneembodiment, the compositions and methods described herein are used totreat a cancer selected from the group consisting of Salivary GlandCancer, Sarcoma, Sezary Syndrome, Skin Cancer, Ocular Cancer, SkinCarcinoma, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous CellCarcinoma, Squamous Neck Cancer with Occult Primary, and SupratentorialPrimitive Neuroectodermal Tumors. In one embodiment, the compositionsand methods described herein are used to treat a cancer selected fromthe group consisting of T-Cell Lymphoma, Testicular Cancer, ThroatCancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional CellCancer of the Renal Pelvis and Ureter, and Gestational TrophoblasticTumor. In one embodiment, the compositions and methods described hereinare used to treat a cancer selected from the group consisting ofCarcinoma of Unknown Primary Site, Cancer of Unknown Primary Site,Unusual Cancers of Childhood, Transitional Cell Cancer of the RenalPelvis and Ureter, Urethral Cancer, and Uterine Sarcoma. In oneembodiment, the compositions and methods described herein are used totreat cancer selected from the group consisting of Vaginal Cancer andVulvar Cancer. In one embodiment, the compositions and methods describedherein are used to treat a cancer selected from the group consisting ofWilms Tumor and Women's Cancers.

In one embodiment, the compositions and methods described herein areused as a first-line therapy (sometimes called primary therapy). In oneembodiment, the compositions and methods described herein are used as asecond-line therapy. In one embodiment, the compositions and methodsdescribed herein are used as a third-line therapy. In one embodiment,the compositions and methods described herein are used as a salvagetherapy. The term “salvage therapy” means a therapeutic agent that canbe taken with any regimen after a subject's initial treatment regimenhas failed or after the subject's condition has not responded to aninitial treatment. In one embodiment, the compositions and methodsdescribed herein are used as a rescue therapy. In some embodiments, thecompositions are used as a rescue agent to counteract the action of aninitial treatment. In some embodiments, the compositions are used asrescue agent which is administered to a subject who has developedresistance to a standard or an initial treatment. In one embodiment, thecompositions and methods described herein are used as a neoadjuvanttherapy. In some embodiments, a neoadjuvant therapy comprisesadministration of one or more of the therapeutic agents described hereinto a subject before a main or first line treatment. In one embodiment, aneoadjuvant therapy reduces the size or extent of the cancer beingtreated before a main or first line treatment is administered to thesubject undergoing treatment. In one embodiment, the compositions andmethods described herein are used as an adjuvant therapy. In oneembodiment, an adjuvant therapy comprises administration of one or moretherapeutic agents described herein to a subject, wherein the one ormore therapeutic agent that modify the effect of other therapeuticagents that are already administered to the subject or are concurrentlyadministered to the subject or subsequently administered to the subject.

In one embodiment, the compositions and methods described herein exhibitreduced chance of drug-drug interactions. In one embodiment, animipridone, such as ONC201, or an analog thereof are eliminated from thepatient's body before it can interact with another pharmaceuticallyactive agent.

In one embodiment, the compositions and methods of described hereinexhibit toxicity levels that facilitates combinations with otherpharmaceutical agents.

The methods and compositions described herein are not limited to aparticular animal species. In one embodiment, a subject treatedaccording to methods and using compositions described herein, can bemammalian or non-mammalian. In one embodiment, a mammalian subjectincludes, but is not limited to, a human; a non-human primate; a rodentsuch as a mouse, rat, or guinea pig; a domesticated pet such as a cat ordog; a horse, cow, pig, sheep, goat, or rabbit. In one embodiment, anon-mammalian subject includes, but is not limited to, a bird such as aduck, goose, chicken, or turkey. In one embodiment, the subject is ahuman. In one embodiment, subjects can be either gender and any age. Thecomposition and methods can also be used to prevent cancer. Thecomposition and methods can also be used to stimulate the immune system.

The methods and compositions described herein are not limited to aparticular age of the subject. In one embodiment, a subject treatedaccording to methods and using compositions described herein is over 50years old, over 55 years old, over 60 years old, or over 65 years old.In one embodiment, a subject treated according to methods and usingcompositions described herein is under 50 years old, under 55 years old,under 60 years old, or under 65 years old.

In one embodiment, a subject treated according to methods and usingcompositions described herein is a pediatric patient. In one embodiment,the pediatric patient is younger than 18 years old, younger than 17years old, younger than 16 years old, younger than 15 years old, youngerthan 14 years old, is younger than 13 years old, younger than 12 yearsold, younger than 11 years old, younger than 10 years old, younger than9 years old, younger than 8 years old, younger than 7 years old, youngerthan 6 years old, younger than 5 years old, younger than 4 years old,younger than 3 years old, younger than 2 years old, younger than 1 yearold. In one embodiment, the pediatric patient is younger than 12 monthsold, younger than 11 months old, younger than 10 months old, youngerthan 9 months old, younger than 8 months old, younger than 7 months old,younger than 6 months old, is younger than 5 months old, younger than 4months old, younger than 3 months old, younger than 2 months old,younger than 1 month old. In one embodiment, the pediatric patientyounger than 4 weeks old, younger than 3 weeks old, younger than 2 weeksold, younger than 1 weeks old. In one embodiment, the pediatric patientis younger than 7 days old, younger than 6 days old, younger than 5 daysold, younger than 4 days old, younger than 3 days old, younger than 2days old, or younger than 1 day old. In one embodiment, the pediatricpatient is a neonate. In one embodiment, the pediatric patient isprematurely born.

In some embodiments, the patient is less than 45 kg in weight, less than40 kg in weight, less than 35 kg in weight, less than 30 kg in weight,less than 25 kg in weight, less than 20 kg in weight, less than 15 kg inweight, less than 14 kg in weight, less than 10 kg in weight, less than5 kg in weight, less than 4 kg in weight, less than 3 kg in weight, lessthan 2 kg in weight, or less than 1 kg in weight.

In one embodiment, the subject has received at least one priortherapeutic agent. In one embodiment the subject has received at leasttwo, at least three, or at least four prior therapeutic agents. In oneembodiment the prior therapeutic agent is ibrutinib, bortezomib,carfilzomib, temozolomide, bevacizumab, cyclophosphamide,hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin,rituximab, 5-fluorouracil, oxaliplatin, leucovorin, or lenalidomide.

In one embodiment, the subject has been treated with radiation. In oneembodiment, the subject has been treated with surgery. In oneembodiment, the subject has been treated with adoptive T-cell therapy.

In one embodiment, the cancer no longer responds to treatment withibrutinib, bortezomib, carfilzomib, temozolomide, bevacizumab,cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone,cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin,leucovorin, lenalidomide, radiation, surgery, or a combination thereof.

In one embodiment, the compositions and methods described herein have adose response relation in cancer cells that is different from the doseresponse relation of the same compositions and methods in normal cells.The dose response relation of ONC201 on proliferation and cell death innormal and tumor cells was determined by measuring cell viabilityfollowing treatment with ONC201 at various concentrations for 72 hours.The tumors tested included a human colon cancer cell line (HCT116),breast tumor cell line (MDA-MB-231), and a human primary glioblastomacell line (U87). And the normal cells tested included human foreskinfibroblasts (HFF), human fetal lung fibroblast (MRC-5) cells, and ahuman lung fibroblast cell line (WI-38). Doxorubicin was used as apositive control at 1 μg/mL in normal fibroblasts. Cell viability ofnormal cells tested was at least about 75% at about 1-5 mg/mL of ONC201,whereas viability of tumor cells was significantly lower (e.g., at orbelow 50%) at the same ONC201 concentration. Moreover, as ONC201concentration increased beyond about 5 mg/mL viability of tumor cellsfell to below 25%, whereas viability of normal cells remained at about75%. Cell viability assays in human fetal lung fibroblast (MRC-5) cellswere performed following 72 hour treatment with compound (1) (5 μM) orDMSO and a recovery period in complete drug-free media after treatment.Cell recovery was seen with ONC201, but not with DMSO.

In some embodiments, the compositions and methods described herein areuseful for treating cancer in a subject. In some embodiments, thecompositions and methods described herein are useful for treating cancerin a human subject. In some embodiments, the treatment method comprisesadministering to a subject in need of such treatment, a pharmaceuticallyeffective amount of an imipridone, such as ONC201, or an analog thereof,or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In one embodiment, the treatment method comprises administering to asubject in need of such treatment: (i) a first therapeutic agentincluding an imipridone, such as ONC201, or an analog thereof, or apharmaceutically acceptable salt thereof in combination with (ii) asecond therapeutic agent, wherein the first and the second therapeuticagents are administered either simultaneously or sequentially. Thesecond therapeutic agent can be any suitable therapeutic agent,including a pharmaceutically active agent disclosed herein. Apharmaceutically acceptable ONC201 salt includes the di-hydrochloridesalt below:

It is understood that a di-hydrochloride salt of ONC201 or an analogthereof (including a compound of formula (10)), or an alternativedi-salt thereof apparent from the teaching of this disclosure, can besubstituted for ONC201 or an analog thereof in a composition or dosingregimen described herein.

In one embodiment, the treatment method comprises administering asynergistic pharmaceutical combination, either simultaneously orsequentially, to a subject in need of such treatment, wherein thesynergistic pharmaceutical combination comprises (i) a first therapeuticagent comprising an imipridone, such as ONC201, or an analog thereof, ora pharmaceutically acceptable salt thereof; and (ii) a secondtherapeutic agent. In one embodiment, the treatment method comprisesadministering to a subject in need of such treatment, eithersimultaneously or sequentially, therapeutically synergistic effectiveamounts of the first therapeutic agent in combination with the secondtherapeutic agent. In one embodiment, the treatment method comprisesadministering to a subject in need of such treatment, an effectiveamount of the first therapeutic agent in combination with an effectiveamount of the second therapeutic agent, wherein the combination providesa synergistic effect in the in vivo treatment of a cancer sensitive tothe combination, and wherein the first and the second therapeutic agentsare administered either simultaneously or sequentially. In oneembodiment, the treatment method comprises administering to a subject inneed of such treatment, an effective amount of the first therapeuticagent in combination with an effective amount of a second therapeuticagent, wherein the combination provides a synergistic effect in the invivo treatment of a minimal residual disease sensitive to thecombination, and wherein the first and second therapeutic agents areadministered either simultaneously or sequentially. In one embodiment,the second therapeutic agent is given before or prior to the firsttherapeutic agent.

In one embodiment, the treatment method targets a cancer selected fromthe group consisting of solid tumors, liquid tumors, lymphomas,leukemias, or myelomas.

In one embodiment, the treatment method targets a solid tumor, whereinthe solid tumor is selected from the group consisting of: CervicalCancer, Endometrial Cancer, Extracranial Germ Cell Tumor; ExtragonadalGerm Cell Tumor; Germ Cell Tumor; Gestational Trophoblastic Tumor;Ovarian Cancer, Ovarian Germ Cell Tumor, Ovarian Epithelial Cancer, andOvarian Low Malignant Potential Tumor; Penile Cancer, Prostate Cancer;Pregnancy and Breast Cancer; high grade prostate cancer; medium gradeprostate cancer; low grade prostate cancer; castration-resistantprostate cancer; Breast Cancer; Bile Duct Cancer; Extrahepatic Bile DuctCancer; Gallbladder Cancer; Hepatocellular (Liver) Cancer; Kidney (RenalCell) Cancer; Liver Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis andUreter; Basal Cell Carcinoma; Basal Cell Nevus Syndrome, Gorlin-NevusSyndrome, Melanoma, Merkel Cell Carcinoma, Papillomatosis, MultipleEndocrine Neoplasia Syndrome; Pancreatic Cancer, Parathyroid Cancer,ocular melanoma; Eye Cancer; Retinoblastoma; Malignant FibrousHistiocytoma; Ewing Sarcoma Family of Tumors; desmoplastic round celltumor; chondrosarcoma, Kaposi Sarcoma, Rhabdomyosarcoma; Spinal CordTumors, Leptomeningeal Disease, Central Nervous System Embryonal Tumors,Chordoma, Embryonal Tumors of Central Nervous System, Ependymoblastoma,Ependymoma, Neuroblastoma; Pineal Parenchymal Tumors of IntermediateDifferentiation, Pineoblastoma; Adrenocortical Carcinoma; Bone Cancer,Osteosarcoma; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma;Osteosarcoma and Malignant Fibrous Histiocytoma of Bone; CarcinoidTumor, Carcinoma of Unknown Primary, Bronchial Tumors, Lung Cancer,Pleuropulmonary Blastoma; Respiratory Tract Carcinoma Involving the NUTGene on Chromosome 15, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor;Central Nervous System Atypical Teratoid/Rhabdoid Tumor,Craniopharyngioma, Glioma, Brain cancer, Medulloblastoma,Medulloepithelioma, Supratentorial Primitive Neuroectodermal Tumors;Pituitary Tumor; Gastric (Stomach) Cancer, Gastrointestinal CarcinoidTumor, Gastrointestinal Stromal Tumor (GIST), Bladder Cancer, Anal orRectal Cancer, Appendix Cancer, Esophageal Cancer, HypopharyngealCancer; Laryngeal Cancer, Lip and Oral Cavity Cancer, MetastaticSquamous Neck Cancer with Occult Primary, Mouth Cancer, Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Oral Cancer, Lip and OralCavity Cancer, Oropharyngeal Cancer, Paranasal Sinus and Nasal CavityCancer, Pharyngeal Cancer; Head and Neck Cancer, and Mesothelioma.

In one embodiment, the treatment method targets a lymphoma selected fromthe group consisting of: diffuse large B-cell lymphoma, AIDS-RelatedLymphoma, Cutaneous T-Cell Lymphoma, Sezary syndrome, mycosis fungoides(MF); Histiocytosis; Burkitt Lymphoma, and Central Nervous SystemLymphoma; Non-Hodgkin Lymphoma, and Primary Central Nervous SystemLymphoma, Hodgkin Lymphoma, Waldenström's macroglobulinemia; MycosisFungoides; Primary Central Nervous System Lymphoma; lymphoplasmacyticlymphoma, and Primary Central Nervous System Lymphoma.

In one embodiment, the treatment method targets a Non-Hodgkin's lymphoma(NHL) selected from the group consisting of: mantle cell lymphoma,diffuse large B-cell lymphoma, follicular lymphoma, marginal zonelymphoma, small lymphocytic lymphoma, lyphoplasmacytic NHL,Waldenstrom's macroglobulinaemia, and skin lymphomas.

In one embodiment, the treatment method targets a leukemia selected fromthe group consisting of: Acute Lymphoblastic Leukemia (ALL), ChronicLymphocytic Leukemia (CLL), Chronic Myeloproliferative Disorders; HairyCell Leukemia; Acute Myeloid Leukemia (AML); Chronic MyelogenousLeukemia (CML); and Langerhans Cell Histiocytosis.

In one embodiment, the treatment method targets an acute leukemiaselected from the group consisting of: acute lymphotyte leukemia, acutemyeloid leukemia, chronic lymphoblasitc leukemia, chronic myeloidleukemia, myelodysplastic syndrome, and myeloproliferative disease.

In one embodiment, the treatment method targets a myeloma selected fromthe group consisting of: IgA myeloma; IgG myeloma; IgM myeloma; IgDmyeloma; IgE myeloma; light chain myeloma; non secretory myeloma;complex karyotype, blastic phase leukemia; Multiple Myeloma/Plasma CellNeoplasm, Multiple Myeloma, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Neoplasms, and MyeloproliferativeDisorders.

In one embodiment, the treatment method targets a peripheral nervoussystem tumor. In one embodiment, the treatment method targets aparaganglioma. In one embodiment, the treatment method targets apheochromocytoma.

In one embodiment, treatment of cancer comprises prevention of tumorgrowth in a cancer subject. In one embodiment, treatment of cancercomprises prevention of formation of cancer metastases in a cancersubject. In one embodiment, treatment of cancer comprises targetedtreatment of minimal residual disease in a cancer subject known to havethe minimal residual disease in a cancer or a subject at risk for havingminimal residual disease.

This might be indicated after treatment of the primary tumor by surgeryand/or after chemotherapy (radiotherapy) has been initiated ordetermined to be efficaceous. Disseminated tumor cells may be in theirdormant state and often cannot be attacked by chemotherapy(radiotherapy). A thus treated patient seemingly is in a healed state,and referred to as “minimal residual disease.” Nevertheless, the dormanttumor cells have a potential to form metastases if they becomemetastasising cells due to a growth stimulus after a longer dormantstate.

The term “minimal residual disease” denotes a small number of cancercells that remain in a subject during or after treatment when thesubject is in remission (exhibiting no symptoms or signs of thedisease). The methods described herein are preferably applied to a formof the diseases listed herein, including adult and childhood forms ofthese diseases.

In one embodiment, the treatment method is useful for treating anautoimmune disease. Autoimmune diseases include, but are not limited toalopecia areata, antiphospholipid, autoimmune hepatits, celiac disease,diabetes type 1, Graves' disease, Guillain-Barre syndrome, Hashimoto'sdisease, hemolytic anemia, idiopathic thrombocytopenic purpura,inflammatory bowel disease, inflammatory myopathies, multiple sclerosis,primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma,Sjögren's syndrome, systemic lupus erythematosus, and vitiligo.

In one embodiment, the treatment method is useful for treatingautoimmune and inflammatory disorders of the peripheral nerve systemsuch as amyotrophic lateral sclerosis (Lou Gehrig's disease), based onvarious causes such as metabolic disorders that include diabetes, B12and folate vitamin deficiencies, chemotherapy medications and medicinesused to treat HIV, poisons that cause peripheral nerve damage, cancersthat develop peripheral neuropathies as well as paraneoplasticsyndromes, alcohol abuse, chronic kidney disease, injuries that causecompression on nerves and other lesions, infections such as Lymedisease, Guillain Barre syndrome, connective tissue disease, rheumatoidarthritis, Sjogren's syndrome, systemic lupus erythematosus, certaininflammatory conditions such as sarcoidosis, coeliac disease, hereditarydiseases such as charcot marie tooth syndrome, Friedreich's ataxia,and/or idiopathic where no specific cause is found but inflammatoryand/or autoimmune mechanisms are the cause of onset.

In one embodiment, the treatment method is useful for treatingautoimmune and inflammatory disorders with ocular manifestations. Suchocular manifestations include, but are not limited to, ocularcicatricial pemphigoid, Mooren's corneal ulcer, various forms ofuveitis, rheumatoid arthritis, systemic lupus erythematosus,polyarteritis nodosa, relapsing polychondritis, Wegener'sgranulomatosis, scleroderma, Behcet's disease, Reiter's disease,inflammatory bowel disease (ulcerative colitis and Crohn's disease) andankylosing spondylitis, retinitis pigmentosa, macular degeneration,keratoconjunctivitis sicca, scleritis, episcleritis, keratitis,peripheral corneal ulceration, and less common entities such aschoroiditis, retinal vasculitis, episcleral nodules, retinaldetachments, and/or macular edema.

In one embodiment, the treatment method is useful for treating acuteallograft rejection in transplant patients. In one embodiment, thetreatment method is useful for treating ischemic stroke. In oneembodiment, the treatment method is useful for treating inflammatorydiseases including arthritis, psoriasis, asthma, and colitis.

In one embodiment, a therapeutic agent includes a pharmaceuticallyacceptable mono-salt of ONC201 or an analog thereof (e.g., a compound offormula (10)). In one embodiment, a therapeutic agent includes apharmaceutically acceptable ONC201 di-salt or an analog thereof (e.g., acompound of formula (10)). As described herein, some of the analogs canbe tri-salts In one embodiment, a therapeutic agent includes ONC201 oran analog thereof (e.g., a compound of formula (10)) in the form of apharmaceutically acceptable mono- or di-salt selected from the groupconsisting of hydrochloride, hydrobromide, hydrogensulphate, sulfates,phosphates, fumarates, succinates, oxalates and lactates, bisulfates,hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate, malate,maleate, fumarate sulfonate, methylsulfonate, formate, acetate, andcarboxylate. In one embodiment, a therapeutic agent includes ONC201 oran analog thereof in the form of a pharmaceutically acceptable mono- ordi-salt selected from p-toluene-sulfonate, benzenesulfonate,methanesulfonate, oxalate, succinate, tartrate, citrate, fumarate andmaleate. In one embodiment, a therapeutic agent includes ONC201 or ananalog thereof in the form of a pharmaceutically acceptable mono- ordi-salt having a counter ion selected from the group consisting ofammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/orwith counter-ions such as methylamino, dimethylamino, diethylamino,triethylamino counter-ions, and combinations thereof. In one embodiment,a therapeutic agent includes a compound described herein in the form ofa halide di-salt, such as a di-hydrochloride salt or a di-hydrobromidesalt.

In some embodiments, the second therapeutic agent includes ananti-cancer agent. In some embodiments, the second therapeutic agent isselected from acivicin, aclarubicin, acodazole, acronine, adozelesin,aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin,ametantrone, amifostine, aminoglutethimide, amsacrine, anastrozole,anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine,azetepa, azotomycin, batimastat, benzodepa, bevacizumab, bicalutamide,bisantrene, bisnafide dimesylate, bizelesin, bleomycin, brequinar,bropirimine, busulfan, cactinomycin, calusterone, capecitabine,caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin,cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine,crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin, decitabine, dexormaplatin, dezaguanine,dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, droloxifene,dromostanolone, duazomycin, edatrexate, eflomithine, elsamitrucin,enloplatin, enpromate, epipropidine, epirubicin, erbulozole, esorubicin,estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine,fenretinide, floxuridine, fludarabine, fluorouracil, flurocitabine,fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea,idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2, includingrecombinant interleukin II or rIL2), interferon α-2a, interferon α-2b,interferon α-n1, interferon α-n3, interferon β-Ia, interferon gamma-Ib,iproplatin, irinotecan, lanreotide, letrozole, leuprolide, liarozole,lometrexol, lomustine, losoxantrone, masoprocol, maytansine,mechlorethamine hydrochlride, megestrol, melengestrol acetate,melphalan, menogaril, mercaptopurine, methotrexate, metoprine,meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid,nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel,pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide,pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin,plomestane, porfimer, porfiromycin, prednimustine, procarbazine,puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine,simtrazene, sparfosate, sparsomycin, spirogermanium, spiromustine,spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin,tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide,teroxirone, testolactone, thiamiprine, thioguanine, thiotepa,tiazofurin, tirapazamine, topotecan, toremifene, trestolone,triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard,uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate,vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine,vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate,zorubicin and combinations thereof.

In some embodiments, the second therapeutic agent is selected, fromhormone analogs and antihormones, aromatase inhibitors, LHRH agonistsand antagonists, inhibitors of growth factors, growth factor antibodies,growth factor receptor antibodies, tyrosine kinase inhibitors;antimetabolites; antitumour antibiotics; platinum derivatives;alkylation agents; antimitotic agents; tubuline inhibitors; PARPinhibitors, topoisomerase inhibitors, serine/threonine kinaseinhibitors, tyrosine kinase inhibitors, protein protein interactioninhibitors, MEK inhibitors, ERK inhibitors, IGF-1R inhibitors, ErbBreceptor inhibitors, rapamycin analogs, amifostin, anagrelid, clodronat,filgrastin, interferon, interferon α, leucovorin, rituximab,procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer,2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyoestradiol,2C4,3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin,16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin,alitretinoin, allovectin-7, altretamine, alvocidib, amonafide,anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, aranose,arglabin, arzoxifene, atamestane, atrasentan, auristatin PE, ABT-199(Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479(ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244(selumetinib), ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717,AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib),avastin, AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006,BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodardicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550,BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid,bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib,brostallicin, busulphan, BYL-719, CA-4 prodrug, CA-4, CapCell,calcitriol, canertinib, canfosfamide, capecitabine,carboxyphthalatoplatin, CC₁₋₇₇9, CC-115, CC-223, CEP-701, CEP-751, CBT-1cefixime, ceflatonin, ceftriaxone, celecoxib, celmoleukin, cemadotin,CH4987655/RO-4987655, chlorotrianisene, cilengitide, ciclosporin,CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicin, combretastatinA4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin52, CTP-37, CTLA-4 monoclonal antibodies, CP-461, CV-247,cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine,deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide,desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol,diflomotecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010,E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFRinhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin,epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3,ethynylcytidine, ethynyloestradiol, exatecan, exatecan mesylate,exemestane, exisulind, fenretinide, figitumumab, floxuridine, folicacid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin,gallium maltolate, gefinitib, gemtuzumab, gimatecan, glufosfamide,GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068,GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100-peptidevaccines, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436(dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183,GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine,histamine, homoharringtonine, hyaluronic acid, hydroxyurea,hydroxyprogesterone caproate, ibandronate, ibritumomab, idatrexate,idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12(cixutumumab), immunol, indisulam, interferon α-2a, interferon α-2b,pegylated interferon α-2b, interleukin-2, INK-1117, INK-128, INSM-18,ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B,isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088,conjugated oestrogens, kahalid F, ketoconazole, KW-2170, KW-2450,lobaplatin, leflunomide, lenograstim, leuprolide, leuporelin,lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol,losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide,marimastat, mechloroethamine, MEK inhibitors, MEK-162,methyltestosteron, methylprednisolone, MEDI-573, MEN-10755, MDX-H210,MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin,mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb,rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453,RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan,R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim,satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101,semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747,SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In some embodiments, the second therapeutic agent is selected fromtamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate,flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproteroneacetate, finasteride, buserelin acetate, fludrocortisone,fluoxymesterone, medroxy-progesterone, octreotide, and combinationsthereof. In some embodiments, the second therapeutic agent is selectedfrom LHRH agonists and LHRH antagonists. In one embodiment, a LHRHagonist is selected from goserelin acetate, luprolide acetate,triptorelin pamoate and combinations thereof. In one embodiment, thesecond therapeutic agent includes a LHRH antagonist is selected fromDegarelix, Cetrorelix, Abarelix, Ozarelix, Degarelix combinationsthereof. In some embodiments, the second therapeutic agent includes aninhibitor of a growth factor. In one embodiment, the inhibitor of agrowth factor is selected from inhibitors of: platelet derived growthfactor (PDGF), fibroblast growth factor (FGF), vascular endothelialgrowth factor (VEGF), epidermal growth factor (EGF), insuline-likegrowth factors (IGF), human epidermal growth factor (HER), hepatocytegrowth factor (HGF), and combinations thereof. In one embodiment, thehuman epidermal growth factor (HER) is selected from HER2, HER3, andHER4.

In some embodiments, the second therapeutic agent includes a tyrosinekinase inhibitor. In some embodiments, the tyrosine kinase inhibitor isselected from cetuximab, gefitinib, imatinib, lapatinib and trastuzumab,and combinations thereof. In some embodiments, the second therapeuticagent includes an aromatase inhibitor. In some embodiments, thearomatase inhibitor is selected from anastrozole, letrozole, liarozole,vorozole, exemestane, atamestane, and combinations thereof.

In some embodiments, the second therapeutic agent includes anantimetabolite. In some embodiments, the antimetabolite comprises anantifolate. In some embodiments, the antifolate is selected frommethotrexate, raltitrexed, pyrimidine analogs, and combinations thereof.In some embodiments, the antimetabolite is a pyrimidine analog. In someembodiments, the pyrimidine analog is selected from 5-fluorouracil,capecitabin, gemcitabin, and combination thereof. In some embodiments,the antimetabolite is a purine analog or an adenosine analog. In someembodiments, the purine analog or adenosine analog is selected frommercaptopurine, thioguanine, cladribine and pentostatin, cytarabine,fludarabine, and combinations thereof. In some embodiments, the secondtherapeutic agent includes an antitumour antibiotic. In someembodiments, the antitumor antibiotic is selected from anthracyclins,doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C,bleomycin, dactinomycin, plicamycin, streptozocin and combinationsthereof. In some embodiments, the second therapeutic agent includes aplatinum derivative. In some embodiments, the platinum derivative isselected from cisplatin, oxaliplatin, carboplatin and combinationsthereof. In some embodiments, the second therapeutic agent includes analkylation agent. In some embodiments, the alkylation agent is selectedfrom estramustin, meclorethamine, melphalan, chlorambucil, busulphan,dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas,and combinations thereof. In some embodiments, the second therapeuticagent includes a nitrosourea. In some embodiments, the nitrosourea isselected from carmustin, lomustin, thiotepa, and combinations thereof.In some embodiments, the second therapeutic agent includes anantimitotic agent. In some embodiments, the antimitotic agent isselected from Vinca alkaloids and taxanes. In some embodiments, thetaxane is selected from paclitaxel, docetaxel, and combinations thereof.In some embodiments, the Vinca alkaloids are selected from vinblastine,vindesin, vinorelbin, vincristine, and combinations thereof. In someembodiments, the second therapeutic agent includes a topoisomeraseinhibitor. In some embodiments, the topoisomerase inhibitor is anepipodophyllotoxin. In some embodiments, the topoisomerase inhibitor,which is an epipodophyllotoxin selected from etoposide, etopophos,teniposide, amsacrin, topotecan, irinotecan, mitoxantron, andcombinations thereof. In some embodiments, the second therapeutic agentincludes a serine/threonine kinase inhibitor. In some embodiments, theserine/threonine kinase inhibitor is selected from PDK 1 inhibitors,B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors,dual mTOR/PI3K inhibitors, STK 33 inhibitors, AKT inhibitors, PLK 1inhibitors, inhibitors of CDKs, Aurora kinase inhibitors, andcombinations thereof. In some embodiments, the second therapeutic agentincludes a tyrosine kinase inhibitor. In some embodiments, the secondtherapeutic agent includes a PTK2/FAK inhibitor. In some embodiments,the second therapeutic agent includes a protein protein interactioninhibitor. In some embodiments, the protein protein interactioninhibitor is selected from IAP, Mcl-1, MDM2/MDMX and combinationsthereof. In some embodiments, the second therapeutic agent includes arapamycin analog. In some embodiments, the rapamycin analog is selectedfrom everolimus, temsirolimus, ridaforolimus, sirolimus, andcombinations thereof. In some embodiments, the second therapeutic agentis selected from amifostin, anagrelid, clodronat, filgrastin,interferon, interferon α, leucovorin, rituximab, procarbazine,levamisole, mesna, mitotane, pamidronate and porfimer, and combinationsthereof. In some embodiments, the second therapeutic agent is selectedfrom 2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine,2-methoxyoestradiol, 2C4,3-alethine, 131-1-TM-601, 3CPA,7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A 105972, A 204197,abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine,alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone,apomine, aranose, arglabin, arzoxifene, atamestane, atrasentan,auristatin PE, ABT-199 (Venetoclax), ABT-263 (Navitoclax), AVLB,AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300,ARRY-142886/AZD-6244 (selumetinib), ARRY-704/AZD-8330, AR-12, AR-42,AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680,AS-703026 (primasertib), avastin, AZD-2014, azacytidine, azaepothiloneB, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab,BEZ-235, biricodar dicitrate, BCX-1777, BKM-120, bleocin, BLP-25,BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807,BNP-1350, BNP-7787, BIBW 2992 (afatinib, tomtovok), BIBF 1120(vargatef), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI853520, BUB-022, bleomycinic acid, bleomycin A, bleomycin B, brivanib,bryostatin-1, bortezomib, brostallicin, busulphan, BYL-719, CA-4prodrug, CA-4, CapCell, calcitriol, canertinib, canfosfamide,capecitabine, carboxyphthalatoplatin, CC₁₋₇₇9, CC-115, CC-223, CEP-701,CEP-751, CBT-1 cefixime, ceflatonin, ceftriaxone, celecoxib,celmoleukin, cemadotin, CH4987655/RO-4987655, chlorotrianisene,cilengitide, ciclosporin, CDA-II, CDC-394, CKD-602, CKI-27, clofarabin,colchicin, combretastatin A4, COT inhibitors, CHS-828, CH-5132799,CLL-Thera, CMT-3 cryptophycin 52, CTP-37, CTLA-4 monoclonal antibodies,CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D 24851,decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide,desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol,diflomotecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010,E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFRinhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin,epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3,ethynylcytidine, ethynyloestradiol, exatecan, exatecan mesylate,exemestane, exisulind, fenretinide, figitumumab, floxuridine, folicacid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin,gallium maltolate, gefinitib, gemtuzumab, gimatecan, glufosfamide,GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068,GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100-peptidevaccines, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436(dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183,GSK-2141795, GW2016, granisetron, herceptine, hexamethylmelamine,histamine, homoharringtonine, hyaluronic acid, hydroxyurea,hydroxyprogesterone caproate, ibandronate, ibritumomab, idatrexate,idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12(cixutumumab), immunol, indisulam, interferon α-2a, interferon α-2b,pegylated interferon α-2b, interleukin-2, INK-1117, INK-128, INSM-18,ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B,isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088,conjugated oestrogens, kahalid F, ketoconazole, KW-2170, KW-2450,lobaplatin, leflunomide, lenograstim, leuprolide, leuporelin,lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol,losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide,marimastat, mechloroethamine, MEK inhibitors, MEK-162,methyltestosteron, methylprednisolone, MEDI-573, MEN-10755, MDX-H210,MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin,mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf ingadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar,neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin,N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage,oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906(linsitinib), 4-1BB antibodies, oxantrazole, oestrogen, panitumumab,patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204,PD0325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilizedpaclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427,P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine,perillylalcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors,PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054,PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate,pixantrone, phenoxodiol 0, PKI166, plevitrexed, plicamycin, polyprenicacid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin,R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436,rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors,revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb,rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453,RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan,R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim,satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101,semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747,SR-13668, SRL-172, sorafenib, spiroplatin, squalamine,suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103,tacedinaline, talaporf in, Tarceva, tariquitar, tasisulam, taxotere,taxoprexin, tazarotene, tegafur, temozolamide, tesmilifene,testosterone, testosterone propionate, tesmilifene, tetraplatin,tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin,thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine,tocladesine, tomudex, toremofin, trabectedin, TransMID-107, transretinicacid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine,triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin,valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1,WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281,XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474,ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat,zosuquidar, and combinations thereof.

In one embodiment, the other therapeutic agent comprises a steroid,including dexamethasone, prednisolone, methyl prednisolone, prednisone,hydrocortisone, triamcinolone, betamethasone, and cortivazol. In oneembodiment, the other therapeutic agent comprises an anti-emetic.Anti-emetics include, but are not limited to, 5-HT3 receptor agonists(such as dolasetron, granisetron, ondansetron, tropisetron,palonosetron, and mirtazapine), dopamine agonists (such as domperidone,olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine,alizapride, prochlorperazine, and metoclopramide), NK1 receptorantagonists (such as aprepitant and casopitant), antihistamines (such ascyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine,promethazine, hydroxyzine), cannabinoids (such as cannabis, dronabinol,nabilone, and sativex), benzodiazepines (such as midazolam andlorazepam), anticholinergics (such as hyoscine), trimethobenzamide,ginger, emetrol, propofol, peppermint, muscimol, and ajwain.

Pharmaceutical compositions may be administered to a subject via anysuitable administration route. In one embodiment, the pharmaceuticalcomposition is administered to a subject orally, parenterally,transdermally or transmucosally. In one embodiment, the pharmaceuticalcomposition is administered to a subject parenterally. In oneembodiment, the pharmaceutical composition is administered to a subjectvia a parenteral administration route selected from intravenous (IV),subcutaneous (SC), and intramuscular (IM). In one embodiment, thepharmaceutical composition is administered to a subject via a route ofadministration selected from rectal and transdermal. In one embodiment,the pharmaceutical composition is administered to a subject in a dosageform selected from the group consisting of sterile solutions,suspensions, suppositories, tablets and capsules. In one embodiment, thepharmaceutical composition is administered to a subject in an oraldosage form selected from the group consisting of a tablet, caplet,capsule, lozenge, syrup, liquid, suspension and elixir. In oneembodiment, the pharmaceutical composition is administered to a subjectin an oral dosage form selected from the group consisting of tablets,hard shell capsules, soft gelatin capsules, beads, granules, aggregates,powders, gels, solids and semi-solids.

In some embodiments, the pharmaceutical composition is administered to asubject as a dosage form selected from sustained release, controlledrelease, delayed release and response release forms.

In one embodiment, the pharmaceutical composition is administered to asubject once daily. In one embodiment, the pharmaceutical composition isadministered to a subject according to an infrequent dosing regimen(e.g., administered once per week or less frequently). In oneembodiment, the pharmaceutical composition is administered to a subjectaccording to a frequent dosing regimen (e.g., administered more thanonce per week). In one embodiment, the pharmaceutical composition isadministered to a subject once weekly. In one embodiment, thepharmaceutical composition is administered to a subject once every fourweeks. In one embodiment, the pharmaceutical composition is administeredto a subject twice a week. In one embodiment, the pharmaceuticalcomposition is administered to a subject once every two weeks. In oneembodiment, the pharmaceutical composition is administered to a subjectonce every three weeks. In one embodiment, the pharmaceuticalcomposition is administered to a subject in a repeated cycle of onceweekly, once every two weeks, once every three weeks, once every fourweeks or combinations thereof.

In one embodiment, the treatment method comprises administering to asubject in need of such treatment: (i) a first therapeutic agentincluding a compound comprising an imipridone, such as ONC201, or ananalog thereof, or a pharmaceutically acceptable salt thereof incombination with (ii) a second therapeutic agent, wherein the firsttherapeutic agent and the second therapeutic agent are administeredeither simultaneously or sequentially; and further comprises assayingthe expression of an endoplasmic reticulum (ER) stress response gene ina biological sample. In one embodiment, the endoplasmic reticulum stressresponse gene is selected from the group that includes, but is notlimited to, C/EBP-Homologous Protein (CHOP), Activating TranscriptionFactor 3 (ATF3) and both CHOP and ATF3. In one embodiment, theendoplasmic reticulum stress response gene is selected from the groupthat includes, but is not limited to, ATF3, Activating TranscriptionFactor 4 (ATF4) CHOP, IRE1, Binding immunoglobulin protein (BiP),Eukaryotic translation initiation factor 2A (eIF2a), X-box bindingprotein 1 (XBP1). The biological sample may be tumor, peripheral bloodmononuclear cells, or skin biopsy. The biological sample may be obtainedbefore, during, or after drug administration. In one embodiment, thetreatment method further comprises adjusting a dose of the firsttherapeutic agent to achieve induction of about 50%, 75%, 100%, 125%,150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%,450%, 475%, 500%, 525%, 550%, 575%, 600%, or greater than 600% of one ormore ER stress gene. In one embodiment, the treatment method furthercomprises adjusting a dose of the first therapeutic agent to achieveinduction of about 50% to about 100%, about 100% to about 150%, about150% to about 200%, about 200% to about 250%, about 250% to about 300%,about 300% to about 350%, about 350% to about 400%, about 400% to about450%, about 450% to about 500%, about 500% to about 550%, about 550% toabout 600%, or greater than 600% of ER stress genes. In one embodiment,the treatment method further comprises adjusting a dose of the firsttherapeutic agent to achieve induction of about 50% to about 100%, about100% to about 200%, about 200% to about 300%, about 300% to about 400%,about 400% to about 500%, about 500% to about 600%, or greater than 600%of ER stress genes.

In one embodiment, the treatment method comprises administering to asubject in need of such treatment: (i) a first therapeutic agentincluding a compound comprising an imipridone, such as ONC201, an analogthereof, or a pharmaceutically acceptable salt thereof in combinationwith (ii) a second therapeutic agent, wherein the first therapeuticagent and the second therapeutic agent are administered eithersimultaneously or sequentially; and further comprises assaying theexpression of proteasomal activity in a biological sample. In oneembodiment the proteasomal activity may be chymotrysin-like,trypsin-like, and/or caspase-like activity. In one embodiment, thebiological sample may be tumor, peripheral blood mononuclear cells, orskin cells. The biological sample may be obtained before, during, orafter drug administration. In one embodiment, the treatment methodfurther comprises adjusting the dose to achieve inhibition of about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, or about 100% of the proteasomal activity. In oneembodiment, the treatment method further comprises adjusting the dose toachieve inhibition of at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the proteasomal activity. In oneembodiment, the treatment method further comprises adjusting the dose toachieve inhibition of about 20% to about 30%, about 30% to about 40%,about 40% to about 50%, about 50% to about 60%, about 60% to about 70%,about 70% to about 80%, about 80% to about 90%, or greater than 90% ofthe proteasomal activity.

In an aspect, provided herein are treatment methods, which compriseadministering to a subject in need of such treatment a combination of afirst therapeutic agent including an imipridone, such as ONC201, ananalog thereof, or a pharmaceutically acceptable salt thereof (e.g., adi-salt or tri-salt) and a second therapeutic agent, the methodcomprising:

(i) administering to the subject the first therapeutic agent;

(ii) waiting until a predetermined waiting time has elapsed after thetime of administration of the first therapeutic agent to the subject;and/or until adverse events are resolved or resolving; and

(iii) administering the second therapeutic agent to the subject, whereinthe predetermined waiting time is chosen so as to obtain a delayedtherapeutic effect of the first therapeutic agent without an increasedrisk of possible combined toxic effects of the first and secondtherapeutic agents. In one embodiment, the predetermined waiting time isdetermined based on the clearance rate of the compound of the firsttherapeutic agent or a metabolite thereof. In one embodiment, thepredetermined waiting time is determined by a quantitative assessment ofrenal function and parameters of renal. In one embodiment, thepredetermined waiting time is determined by an assay for thedetermination of renal function, wherein the assay is selected from thegroup consisting of serum level the compound of the first therapeuticagent or a metabolite thereof; clearance rate of the compound of thefirst therapeutic agent or a metabolite thereof; 24-hour urinaryclearance of the compound of the first therapeutic agent or a metabolitethereof.

In some embodiments, the predetermined waiting time substantially equalsthe time required for systemic clearance of the compound of the firsttherapeutic agent or a metabolite thereof from the subject's body. Insome embodiments, the predetermined waiting time substantially equalsthe time required for renal clearance of the compound of the firsttherapeutic agent or a metabolite thereof from the subject's body. Insome embodiments, the predetermined waiting time substantially equalsthe time required for hepatic clearance of the compound of the firsttherapeutic agent or a metabolite thereof from the subject's body. Insome embodiments, the predetermined waiting time substantially equalsthe time required for total clearance of the compound of the firsttherapeutic agent or a metabolite thereof from the subject's body. Insome embodiments, the predetermined waiting time is about 4 hours. Inother embodiments the waiting time is 1 day. In one embodiment, thewaiting time is until C_(max) of the compound of the first therapeuticagent has passed. In other embodiments, the waiting time is after mostof the adverse events are resolved or are resolving. In someembodiments, the predetermined waiting time is about 2 days, about 3days, about 4 days, about 5 days, about 6 days, or about 7 days. In someembodiments, the predetermined waiting time is a range of about 1-7days, about 1-6 days, about 1-5 days, about 1-4 days, about 1-3 days, orabout 1 to 2 days. In one embodiment, the waiting time is up to 3 weeks.The preceeding are considered “therapeutic time periods.”

When the order of administration is reversed, timing for theadministration of the first therapeutic agent can be after the C_(max)of the second therapeutic agent (i.e., the first administered drug) haspassed. In one embodiment, administration of the first therapeutic agentcan be after most or substantially all of the first administered drughas been eliminated from the body or the toxicity effects for the firstadministered drug are resolved or are resolving.

In one embodiment, the treatment method further comprises monitoringlevels of the compound of the first therapeutic agent or a metabolitethereof in the subject using pharmacokinetic profiling. In some suchembodiments, monitoring levels of the compound of the first therapeuticagent or a metabolite thereof in the subject using pharmacokineticprofiling comprises constructing a pharmacokinetic profile of thecompound of the first therapeutic agent or a metabolite thereof for thesubject using concentrations of the compound of the first therapeuticagent or a metabolite thereof in at least two samples obtained from thesubject at time points suitable to construct a pharmacokinetic profile.In one embodiment, which include monitoring levels of the compound ofthe first therapeutic agent or a metabolite thereof in the subject usingpharmacokinetic profiling, samples are collected from the subject atpoint-of-care or point of use by sampling or self-sampling onpoint-of-care devices or point of use devices or on matrices suitablefor storage of the samples prior to quantitation in a laboratory. In oneembodiment, each of the point-of-care devices or point of use devices iscapable of quantitating the compound of the first therapeutic agent or ametabolite thereof. In one embodiment, which include monitoring levelsof the compound of the first therapeutic agent or a metabolite thereofin the subject, one or more samples are collected from the subject atpoint-of-care or point of use by biopsy device for analysis at thepoint-of-care or point of use devices or for storage prior to analysisby a laboratory. In one embodiment, a biopsy is taken after a timeinterval of 3-8 hours following administration the first therapeuticagent to the subject. In one embodiment, a biopsy is taken after a timeinterval of 3-24 hours following administration of the first therapeuticagent to the subject. In one embodiment, a biopsy is taken after a timeinterval of 8-24 hours following administration of the first therapeuticagent thereof to the subject. In one embodiment, a biopsy is taken aftera time interval of 2 days following administration of the firsttherapeutic agent to the subject. In one embodiment, a biopsy is takenafter a time interval of 3 days following administration of the firsttherapeutic agent to the subject. In one embodiment, a biopsy is takenafter a time interval of 4 days following administration of the firsttherapeutic agent to the subject. In one embodiment, a biopsy is takenafter a time interval of 1-7 days following administration of the firsttherapeutic agent.

In one embodiment, the pharmacokinetic profile includes pharmacokineticparameters suitable for guiding dosing of the first therapeutic agentfor the subject being treated. In some embodiments, the C_(max) of thefirst therapeutic agent following its administration to the subjectranges from about 1000 ng/dL to 1500 ng/dL for a therapeutic timeperiod. In one embodiment, C_(max) is less than 1500 ng/dL and greaterthan 85 ng/dL for a therapeutic time period. In one embodiment, theC_(max) of the first therapeutic following its administration to thesubject ranges from about 1000 ng/mL to 1500 ng/mL for a therapeutictime period. In one embodiment, C_(max) is less than 1500 ng/mL andgreater than 85 ng/mL for a therapeutic time period.

In one embodiment, maximum concentration of the first therapeutic agentin blood (whole blood, plasma, or serum) (“C_(max)”) of a subject afteradministering it to the subject is a C_(max) of from about 1000, 1010,1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130,1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250,1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370,1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or1490 ng/dL to about 1500 ng/dL; from about 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, or 149 ng/dL to about 150 ng/dL; or from about 10, 10.5, 11, 11.5,120, 12.5, 13, 13.5, 14, or 14.5 ng/dL to about 15 ng/dL.

In one embodiment, maximum concentration of the first therapeutic agentin blood (whole blood, plasma, or serum) (“C_(max)”) of the subjectfollowing its administration is a C_(max) of from about 1000, 1010,1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130,1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250,1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370,1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or1490 ng/mL to about 1500 ng/mL; from about 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, or 149 ng/mL to about 150 ng/mL; or from about 10, 10.5, 11, 11.5,120, 12.5, 13, 13.5, 14, or 14.5 ng/mL to about 15 ng/mL.

In one embodiment, maximum concentration of the first therapeutic agentin blood (whole blood, plasma, or serum) (“C_(max)”) of a subjectfollowing its administration is selected from about 1000, 1010, 1020,1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140,1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260,1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380,1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or 1490ng/dL. In one embodiment, the C_(max) of the first therapeutic agent inblood (whole blood, plasma, or serum) (“C_(max)”) of a subject followingits administration is selected from about 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, or 149 ng/dL. In one embodiment, the C_(max) of the firsttherapeutic agent following its administration is selected from about10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng/dL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration is selected from about 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160,1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280,1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400,1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or 1490 ng/mL. In oneembodiment, the C_(max) of the first therapeutic agent following itsadministration is selected from about 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or149 ng/mL. In one embodiment, the C_(max) of the first therapeutic agentfollowing its administration is selected from about 10, 10.5, 11, 11.5,120, 12.5, 13, 13.5, 14, or 14.5 ng/mL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration is selected from about 85, 95, 105, 115, 125, 135,145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275,285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415,425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555,565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695,705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835,845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975,985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095,1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215,1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335,1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455,1465, 1475, 1485, 1495, or 1500 ng/dL. In one embodiment, the C_(max) ofthe first therapeutic agent following its administration is selectedfrom about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL. In oneembodiment, the C_(max) of the first therapeutic agent following itsadministration is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, or 14.5 ng/dL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration is selected from about 85, 95, 105, 115, 125, 135,145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275,285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415,425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555,565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695,705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835,845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975,985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095,1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215,1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335,1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455,1465, 1475, 1485, 1495, or 1500 ng/mL. In one embodiment, the C_(max) ofthe first therapeutic following its administration is selected fromabout 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/mL. In oneembodiment, the C_(max) of the first therapeutic agent following itsadministration is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, or 14.5 ng/mL.

In one embodiment, the C_(max) of the first therapeutic agent afteradministering it to the subject ranges from about 85 ng/dL to 1500ng/dL; from about 8.5 ng/dL to 150 ng/dL; or from about 0.85 ng/dL to 15ng/dL. In one embodiment, the C_(max) of the first therapeutic agent ina subject's blood (whole blood, plasma, or serum) after itsadministration is selected from about 85, 95, 105, 115, 125, 135, 145,155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285,295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425,435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565,575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705,715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845,855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985,995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105,1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225,1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345,1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465,1475, 1485, or 1495 ng/dL to about 1500 ng/dL; from about 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, or 149 ng/dL to about 150 ng/dL; or fromabout 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/dL to about15 ng/dL.

In one embodiment, the C_(max) of the first therapeutic agent followingits administration ranges from about 85 ng/mL to 1500 ng/mL; from about8.5 ng/mL to 150 ng/mL; or from about 0.85 ng/mL to 15 ng/mL. In oneembodiment, the C_(max) of the first therapeutic following itsadministration is selected from about 85, 95, 105, 115, 125, 135, 145,155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285,295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425,435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565,575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705,715, 725, 735, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835,845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975,985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095,1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215,1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335,1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455,1465, 1475, 1485, or 1495 ng/mL to about 1500 ng/mL; from about 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, or 149 ng/mL to about 150 ng/mL; orfrom about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/mLto about 15 ng/mL.

In one embodiment, the total drug exposure over time, measured as thearea under the curve (“AUC”) of a plot of the concentration of the drugin blood (whole blood, plasma, or serum) of a subject followingadministration of the drug against time after administration of the drugranges from about 150 ng hr/mL to about 8000 ng hr/mL; from about 15 nghr/mL to about 800 ng hr/mL; or from about 1.5 ng hr/mL to about 80 nghr/mL. In one embodiment, AUC is less than 8000 ng hr/mL and is greaterthan or equal to 150 ng hr/mL. In one embodiment, AUC is less than 800ng hr/mL and is greater than or equal to 15 ng hr/mL. In one embodiment,AUC is less than 80 ng hr/mL and is greater than or equal to 1.5 nghr/mL.

In one embodiment, the total drug exposure over time is an AUC of fromabout 100 ng hr/mL to about 8000 ng hr/mL; from about 10 ng hr/mL toabout 800 ng hr/mL; or from about 1 ng hr/mL to about 80 ng hr/mL. Inone embodiment, the total drug exposure over time is an AUC of fromabout from about 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800,2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600,6800, 7000, 7200, 7400, 7600, or 7800 ng hr/mL to about 8000 ng hr/mL.In one embodiment, the total drug exposure over time is an AUC of fromabout 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540,560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, or 780 ng hr/mLto about 800 ng hr/mL. In one embodiment, the total drug exposure overtime is an AUC of from about from about 1.5, 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78 ng hr/mL to about80 ng hr/mL.

In one embodiment, the total drug exposure over time is an AUC of fromabout 100 ng hr/mL to about 8000 ng hr/mL, from about 10 ng hr/mL toabout 800 ng hr/mL; or from about 1 ng hr/mL to about 80 ng hr/mL. Inone embodiment, the total drug exposure over time is an AUC of fromabout from about 150 ng hr/mL to about 7800, 7600, 7400, 7200, 7000,6800, 6600, 6400, 6200, 6000, 5800, 5600, 5400, 5200, 5000, 4800, 4600,4400, 4200, 4000, 3800, 3600, 3400, 3200, 3000, 2800, 2600, 2400, 2200,2000, 1800, 1600, 1400, 1200, 1000, 800, 600, 400, or 200 ng hr/mL. Inone embodiment, the total drug exposure over time is an AUC of fromabout from about 15 ng hr/mL to about 780, 760, 740, 720, 700, 680, 660,640, 620, 600, 580, 560, 540, 520, 500, 480, 460, 440, 420, 400, 380,360, 340, 320, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100,80, 60, 40, or 20 ng hr/mL. In one embodiment, the total drug exposureover time is an AUC of from about from about 1.5 ng hr/mL to about 78,76, 74, 72, 70, 68, 66, 64, 62, 60, 58, 56, 54, 52, 50, 48, 46, 44, 42,40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4,or 2 ng hr/mL. In one embodiment, the total drug exposure over time isan AUC of from about 100 ng hr/mL to about 200 ng hr/mL; from about 10ng hr/mL to about 20 ng hr/mL; or from about 1 ng hr/mL to about 2 nghr/mL.

In one embodiment, the total drug exposure over time is an AUC selectedfrom about 100, 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800,2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 46000, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600,6800, 7000, 7200, 7400, 7600, 7800, and 8000 ng hr/mL. In oneembodiment, the total drug exposure over time is an AUC selected fromabout 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240,260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 4600, 480, 500, 520,540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, and 800ng hr/mL. In one embodiment, the total drug exposure over time is an AUCselected from about 1, 15, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 460, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, and 80 ng hr/mL.

In another aspect, provided herein are methods of treatment, or use of acomposition to treat a disease state, which comprises administering to asubject in need of such treatment a combination of a first therapeuticagent and a second therapeutic agent, the method comprising:

(i) administering to the subject the first therapeutic agent includingan imipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof;

(ii) monitoring levels of the compound of the first therapeutic agent ora metabolite thereof in the subject using pharmacokinetic profiling; and

(iii) administering the second therapeutic agent conditional on thelevel of the first therapeutic agent in the subject. In one embodiment,the monitoring step includes constructing a pharmacokinetic profile ofthe compound of the first therapeutic agent or a metabolite thereof forthe subject using concentrations of the compound of the firsttherapeutic agent or a metabolite thereof in a plurality of samplesobtained from the subject at time points suitable to construct apharmacokinetic profile. In one embodiment, at least two samples arecollected at point-of-care or point of use by sampling or self-samplingon point-of-care devices or point of use devices or on matrices suitablefor storage of the samples prior to quantitation of the compound or ametabolite thereof by a laboratory. In one embodiment, eachpoint-of-care devices or point of use devices is capable of quantitatingthe compound or a metabolite thereof. In one embodiment, thepharmacokinetic profile includes pharmacokinetic parameters suitable forguiding dosing of the compound or a salt thereof for the subject. In oneembodiment, the samples include from 2-12 samples. In one embodiment,the samples are collected over a time period of up to 8 hours, up to 24hours, up to 48 hours, or up to 72 hours. In one embodiment, thepharmacokinetic parameters include at least one parameter selected fromthe group consisting of AUC, AUC_(inf), T_(max), C_(max), time abovethreshold, steady state concentration, absorption rate, clearance rate,distribution rate, terminal T-1/2 or parameters drawn fromnoncompartmental pharmacokinetic (PK) or compartmental PK analysis,including physiological model-based compartmental PK analysis. In oneembodiment, the treatment method further comprises generating a reportincluding the pharmacokinetic profile of the subject. In one embodiment,the report includes a recommendation regarding dosing based on thepharmacokinetic profile of the subject. In one embodiment, a reductionin dosage of ONC201, the analog thereof, or the pharmaceuticallyacceptable salt thereof is indicated to reduce risk of toxicity based onone or more pharmacokinetic parameters. In one embodiment, the reductionin dosage of the compound or salt thereof is indicated based on timeabove threshold, wherein the threshold is the drug concentration abovewhich toxicity occurs, or one or more of AUC, AUC_(inf), mean residencetime (MRT), exponentials defining the pharmacokinetic profile, volume ofdistribution at steady state (Vss), volume of distribution during theterminal phase (Vz) or combination of a group of pharmacokineticvariable to adequately describe the pharmacokinetic profile. In oneembodiment, a dose adjustment of the compound or salt thereof isindicated to increase efficacy based on one or more pharmacokineticparameters. In one embodiment, an increase in dosage of the compound orsalt thereof is indicated based on one or more of AUC, AUC_(inf), MRT,exponentials defining the pharmacokinetic profile, steady state volume(Vss) of distribution, volume of distribution during the terminal phase(Vz) or combination of a group of pharmacokinetic variables toadequately describe the pharmacokinetic profile. In one embodiment, thedose of the compound or salt thereof is adjusted to within 5% to 25% ofa desired target value. In one embodiment, each of the samples isapplied to the point-of-care device or the point of use device fordetermining the concentration of the compound or a metabolite thereof,wherein the point-of-care device or the point of use device comprises alateral flow strip having a construction and composition such that anapplication of one or more of the samples to the lateral flow stripcauses a fraction of the drug in the sample to bind to with a componentof the lateral flow strip such that a detectable signal proportional tothe concentration of the drug in the applied sample is produced. In oneembodiment, the samples are applied to matrices suitable for storage ofthe samples prior to quantitation by a laboratory. In one embodiment,the samples are stored as dried blood spots. In one embodiment, drugconcentrations are measured by ELISA, LC MS MS, LC UV or LCMS. In oneembodiment, the pharmacokinetic parameters include at least one ofsteady state concentration, absorption, and terminal T₁/2. In oneembodiment, at least one of the samples is whole blood.

IX. Multimodal Therapeutic Methods

In one aspect, provided herein are multimodal therapeutic methods inwhich administration of an imipridone, such as ONC201, an analogthereof, or a pharmaceutically acceptable salt thereof to a subject inneed of such treatment is supplemented by administration of othertherapeutic modalities. In one embodiment, the multimodal therapeuticmethod comprises administering to a subject a pharmaceutical compositioncomprising an imipridone, such as ONC201, an analog thereof, or apharmaceutically acceptable salt thereof in conjunction with radiationtherapy or after radiation is determined to not have been efficacious.In one embodiment, the multimodal therapeutic method comprisesadministering to a subject a pharmaceutical composition comprising animipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof in conjunction with radiation therapy, whereinthe pharmaceutical composition comprising theimipridone, such as ONC201,the analog thereof, or pharmaceutically acceptable salt thereof and theradiation therapy are administered concurrently or sequentially in anyorder. In one embodiment, the multimodal therapeutic method comprisesadministering to a subject a pharmaceutical composition comprising animipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof in conjunction with radiation therapy in asequential arrangement. In one embodiment, the multimodal therapeuticmethod comprises administering to a subject in need of such treatment apharmaceutical composition comprising an imipridone, such as ONC201, ananalog thereof, or a pharmaceutically acceptable salt thereof thereofconcurrently with radiation therapy. In one embodiment, the multimodaltherapeutic method is used for the treatment of cancer. In oneembodiment, the multimodal therapeutic method includes administering toa cancer subject in need of such treatment a pharmaceutical compositioncomprising an imipridone, such as ONC201, an analog thereof, or apharmaceutically acceptable salt thereof and irradiating cancer cellswith a radiation beam. In one embodiment, the multimodal therapeuticmethod uses the technique of conformal radiotherapy (CRT) to deliver adose volume histogram (DVH) prescribed to a cancer subject. In oneembodiment, the multimodal therapeutic method uses the technique ofintensity modulated radiation therapy (IMRT) to deliver radiation tocancer cells. In one embodiment, the multimodal therapeutic method usestechniques that compensate for motion of tumors in the subject duringtreatment (e.g., where doses of radiation must be administered to athoracic tumor which moves as the patient breathes). For example, themultimodal therapeutic method use Four Dimensional Computed Tomography(4D CT) scanning techniques to adjust the delivered radiation field tocompensate for tumor motion over the breathing cycle.

Any suitable type of radiation, including gamma radiation which is givenfractionated, IMRT (intensity modulated radiation therapy), gamma knife,proton therapy and brachytherapy can be used with the multimodaltherapeutic method. Radiation therapy and administering an imipridone,such as ONC201, an analog thereof, or a pharmaceutically acceptable saltthereof can be used to treat brain tumors such as glioblastoma ordisease that has metastasized to the brain from lung cancer. Themultimodal therapeutic method can be used to treat lung cancer,pancreatic cancer, rectal cancer, breast cancer, sarcoma, prostatecancer, gynecological malignancies, and lymphoma. The gamma knife isused frequently to treat brain metastases. In one embodiment, themultimodal therapeutic method includes use of proton therapy to treatcancer, including brain tumors, prostate cancer and any tumor proximatevital organs where it is very important to minimize toxicity to nearbynormal tissue.

In one embodiment, a multimodal therapeutic method includesadministering to a cancer subject in need of such treatment apharmaceutical composition comprising an imipridone, such as ONC201, ananalog thereof, or a pharmaceutically acceptable salt thereof incombination with adoptive cell therapy (e.g., CAR-T (JCAR 14, 15, 16,17, KTE-C19, or CTL019); other T Cell (AFM13); or NK (CDNO-109 orNK-92)) either simultaneously or in combination.

In one embodiment, the multimodal therapeutic method eliminates minimalresidual disease without adding to toxicity resulting from treatment byan imipridone, such as ONC201, an analog thereof, or a pharmaceuticallyacceptable salt thereof. In one embodiment, the multimodal therapeuticmethod improves prognosis and/or reduces adverse side-effects associatedwith a disease state or condition in a subject undergoing treatment.

X. Additional Imipridone Derivatives, Analogs, and Salts

In one aspect, provided herein are compounds that are analogs of thecompounds of formula (10) and methods of making them. Persons skilled inthe art will understand that the general principles and conceptsdescribed above in conjunction with ONC201 and compounds of formula (10)and their salts, including principles and concepts related to methodsand pharmaceutical compositions, apply with equal force to the followinganalogs and salts thereof.

In one embodiment, the analogs have the structure of compound (25):

wherein Y is NR₄ or O, and wherein R₁, R₂, R₃, and R₄ independentlyrepresent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl,alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy,aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl,mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl,alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocycle radicals.In one embodiment, R₁, R₂, R₃, and R₄ are optionally substituted. In oneembodiment, some or all hydrogens in R₁, R₂, R₃, and R₄ are substitutedby deuterium. In other embodiments, R₁, R₂, R₃, and R₄ are independentlyselected from the group consisting of H, C₁₋₄alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, and C₁₋₄alkylthienyl,wherein C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, andC₁₋₄benzyl-piperazine are optionally substituted with C₁₋₄alkyl,hydroxyl, or halo. In still other embodiments, R₁, R₂, R₃, and R₄ areindependently selected from the group consisting of H, CH₃, CH₂Ph,CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph.

In one embodiment, the analogs have the structure of compound (26):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C14alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionallysubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, R₁ is selected from the groupconsisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph.

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₂ is a benzyl substituted withone or more of the following substituents alone or in combination in theortho, meta, and/or para positions of the benzyl ring: —CH₃, —NO₂,—OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2to 20 and where X is a halogen.

In one embodiment, R1 is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from the group consisting of halo,—CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted or anunsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (27):

wherein R₁ is H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl,haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl,alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio,alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl,alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocycle radicals.In one embodiment, R₁ is selected from the group consisting of H,C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone,C₁₋₄benzyl-piperazine, and C₁₋₄alkylthienyl, wherein C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine areoptionally substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, R₁ is selectedfrom the group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph),CH₂-(2-thienyl), CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-diF-Ph), CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph.

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₁ is a H. In one embodiment, R₁is a substituted or an unsubstituted arylalkyl, such as a benzyl orphenylethyl group. In one embodiment, the arylalkyl is substituted withC₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, the analogs have the structure of compound (28):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C14alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionallysubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, R₁ is selected from the groupconsisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph), CH₂CHOHPh,CH₂CH₂(4-N-benzyl-piperazine), and (CH₂)₃CO-4F-Ph. In one embodiment, R₂is selected from the group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph),CH₂-(2-thienyl), CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-diF-Ph), CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In oneembodiment, when R₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In oneembodiment, R₁ is CH₂Ph and R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁is CH₂Ph and R₂ is CH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph andR₂ is CH₂-(4-CF₃-Ph).

In one embodiment, R1 is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₂ is a benzyl substituted withone or more of the following substituents alone or in combination in theortho, meta, and/or para positions of the benzyl ring: —CH₃, —NO₂,—OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2to 20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from the group consisting of halo,—CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted or anunsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (29):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionallysubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, R₁ is selected from the groupconsisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, whenR₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including refers to F, Cl, Br, or I; preferably, F, Cl,or Br; more preferably, F or Cl. In one embodiment, R₂ is a benzylsubstituted with one or more of the following substituents alone or incombination in the ortho, meta, and/or para positions of the benzylring: —CH₃, —NO₂, —OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is aninteger from 2 to 20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C1-4alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from the group consisting of halo,—CH₃, —CF₃, and —OC_(H)3. In one embodiment, R₂ is a substituted or anunsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (30):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionallysubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, R₁ is selected from the groupconsisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, whenR₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including refers to F, Cl, Br, or I, preferably, F, Cl,or Br, more preferably, F or Cl. In one embodiment, R₂ is a benzylsubstituted with one or more of the following substituents alone or incombination in the ortho, meta, and/or para positions of the benzylring: —CH₃, —NO₂, —OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is aninteger from 2 to 20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from the group consisting of halo,—CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted or anunsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted orunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, the analogs have the structure of compound (31):

wherein R₁ and R₂ independently represent H, alkyl, cycloalkyl,cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl,aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl,alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio,arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl,heteroaryl, acyl, and heterocycle radicals. In one embodiment, R₁ and R₂are independently selected from the group consisting of H, C₁₋₄alkyl,C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, andC₁₋₄alkylthienyl, wherein C₁₋₄alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, and C₁₋₄benzyl-piperazine are optionallysubstituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, R₁ is selected from the groupconsisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl), CH₂CH₂Ph,CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph), CH₂-((2-CH₃)-Ph),CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, R₂ is selected fromthe group consisting of H, CH₃, CH₂Ph, CH₂-((2-Cl)-Ph), CH₂-(2-thienyl),CH₂CH₂Ph, CH₂CH₂(4-N-benzyl-piperazine), CH₂-(2,4-di F-Ph),CH₂-((2-CH₃)-Ph), CH₂CHOHPh, and (CH₂)₃CO-4F-Ph. In one embodiment, whenR₁ is CH₂Ph, R₂ is not CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(2,4-di F-Ph). In one embodiment, R₁ is CH₂Ph and R₂ isCH₂-(4-CF₃-Ph).

In one embodiment, R₁ is a benzyl optionally substituted with one ormore of the following substituents alone or in combination in the ortho,meta, and/or para positions of the benzyl ring: —CH₃, —NO₂, —OCH₃,—CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃, —C_(p)X_(2p+1),—OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2 to 20 and whereX is a halogen including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl. In one embodiment, R₂ is a benzyl substituted withone or more of the following substituents alone or in combination in theortho, meta, and/or para positions of the benzyl ring: —CH₃, —NO₂,—OCH₃, —CXH₂, —CX₂H, —CX₃, —CH₂(CX₃), —CH(CX₃)₂, —C(CX₃)₃,—C_(p)X_(2p+1), —OCX₃, or —OC_(p)X_(2p+1), where p is an integer from 2to 20 and where X is a halogen.

In one embodiment, R₁ is a H. In one embodiment, R₁ is a substituted oran unsubstituted arylalkyl, such as a benzyl or phenylethyl group. Inone embodiment, the arylalkyl is substituted with C₁₋₄alkyl,C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, or halo.

In one embodiment, R₂ is a substituted or an unsubstituted arylalkyl,such as a benzyl or phenylethyl group. In one embodiment, the arylalkylis substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenatedC₁₋₄alkyl, or halo. In one embodiment, the arylalkyl is substituted withone or more substituents selected from the group consisting of halo,—CH₃, —CF₃, and —OCH₃. In one embodiment, R₂ is a substituted or anunsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl orpiperazinylalkyl group. In one embodiment, R₂ is a substituted or anunsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl orpyridylmethyl group. In one embodiment, the heterocycloalkylalkyl orheteroarylalkyl is substituted with C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl,perhalogenated C₁₋₄alkyl, or halo. In one embodiment, theheterocycloalkylalkyl or heteroarylalkyl is substituted with one or moresubstituents selected from the group consisting of halo, —CH₃, —CF₃, and—OCH₃.

In one embodiment, provided herein are compounds of formula (100):

wherein R₁ and R₂ are independently selected from H, alkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl,aralkoxy, aralkylthio, and acyl radicals. In one embodiment, R₁ is CH₂Phand R₂ is CH₂-(2-CH₃-Ph), which is an ONC201 linear isomer

TIC-10, which lacks anti-cancer activity (Jacob et al., Angew. Chem.Int. Ed., (2014) 53:6628; Wagner et al., Oncotarget (2015) 5(24):12728).TIC-10 is a CXCR7 agonist. CXCR7 agonists can be used for liverregeneration and to prevent or treat liver fibrosis.

In one embodiment, R₁ and R₂ are independently selected from the groupconsisting of H, C₁₋₄alkyl, C₁₋₄alkylphenyl, C₁₋₄alkylphenylketone,C₁₋₄benzyl-piperazine, C₁₋₄alkylthienyl, C₁₋₄alkylpyridinyl,C₁₋₄alkylisoxazolidinyl, C₁₋₄alkylmorpholinyl, C₁₋₄alkylthiazolyl, andC₁₋₄alkylpyrazinyl wherein C₁₋₄alkyl, C₁₋₄alkylphenyl,C₁₋₄alkylphenylketone, C₁₋₄benzyl-piperazine, C₁₋₄alkylthienyl,C₁₋₄alkylpyridinyl, C₁₋₄alkylisoxazolidinyl, C₁₋₄alkylmorpholinyl,C₁₋₄alkylthiazolyl, and C₁₋₄alkylpyrazinyl are optionally substitutedwith C₁₋₄alkyl, C₁₋₄alkoxyl, hydroxyl, perhalogenated C₁₋₄alkyl, orhalo. In one embodiment, R₁ and/or R₂ is a substituted or unsubstituted,arylalkyl or heteroarylalkyl. In one embodiment, the heteroarylalkyl isselected from C₁₋₄alkylpyrrolyl, C₁₋₄alkylfuryl, C₁₋₄alkylpyridyl,C₁₋₄alkyl-1,2,4-thiadiazolyl, C₁₋₄alkylpyrimidyl, C₁₋₄alkylthienyl,C₁₋₄alkylisothiazolyl, C₁₋₄alkylimidazolyl, C₁₋₄alkyltetrazolyl,C₁₋₄alkylpyrazinyl, C₁₋₄alkylpyrimidyl, C₁₋₄alkylquinolyl,C₁₋₄alkylisoquinolyl, C₁₋₄alkylthiophenyl, C₁₋₄alkylbenzothienyl,C₁₋₄alkylisobenzofuryl, C₁₋₄alkylpyrazolyl, C₁₋₄alkylindolyl,C₁₋₄alkylpurinyl, C₁₋₄alkylcarbazolyl, C₁₋₄alkylbenzimidazolyl, andC₁₋₄alkylisoxazolyl.

In one embodiment, R₁ and/or R₂ is a benzyl optionally substituted withone or more of the following substituents on the benzyl ring: X, —CH₃,—NO₂, —OCH₃, —CN, —CXH₂, —CX₂H, C₂-C₄ alkyl, —CX₃, —CH₂(CX₃), —CH(CX₃)₂,—C(CX₃)₃, —C_(p)X_(2p+1), —OCX₃, —OC_(p)H_(2p+1), —OC_(p)X_(2p+1),OR^(m), SR^(m), NR^(m)R^(n), NR^(m)C(O)R^(n), SOR^(m), SO₂R^(m),C(O)R^(m), and C(O)OR^(m); R^(m) and R^(n) are independently selectedfrom H or a C₁-C₄ alkyl; and where p is an integer from 2 to 20 and X isa halogen, including F, Cl, Br, or I; preferably, F, Cl, or Br; morepreferably, F or Cl.

XI. Examples

It should be understood that the description and examples below aremeant only for illustration purposes and are not meant to limit thescope of this disclosure. The examples below are meant to illustrate theembodiments disclosed and are not to be construed as being limitationsto them. Additional compounds, other than those described below, may bemade by the following reaction schemes or appropriate variations ormodifications thereof.

Example 1. Synthesis of 2-Chlorobenzylamino-2-imidazoline hydriodide

To a stirred solution of 2-methylthio-2-imidazoline hydriodide (244 mg,1.00 mMol) in dry dioxane (2.0 mL) was added 2-chlorobenzylamine (141mg, 1.0 mMol). The reaction mixture was stirred for 90 min at 70° C.under argon. The solution was cooled to room temperature, filtered on asintered funnel, washed with cold dioxane (2 mL) and dried under vacuum.The white solid compound 4.HI (R₂=2-chlorobenzyl) was obtained (242 mg,72%) and used without further purification.

Example 2. Synthesis of 2-Chlorobenzylamino-2-imidazoline

To a stirred solution of 2-chlorobenzylamino-2-imidazoline hydriodide(242 mg, 0.72 mMol) in water (3 mL), was added 1.0 N sodium hydroxide (2mL) at 7° C. The reaction mixture was stirred for 30 min at 7° C. underargon. After that methylene chloride (5 mL) was added and the mixturestirred for another 5 min. The reaction mixture was extracted withmethylene chloride (2×2.5 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered and evaporated. The resulting free base (150mg, 100%) was obtained as a viscous liquid and was used for the nextreaction without any further purification. MS(ESI) 210(M+H).

Example 3. Synthesis of Methyl-1-benzyl 4-oxo-3-piperidine carboxylate(Compound (6))

To a stirred methyl-1-benzyl 4-oxo-3-piperidine carboxylatehydrochloride (5.7 g, 20 mMol) in ethyl acetate (50 mL), was addedtriethylamine (6 mL) at 7° C. The reaction mixture was stirred for 30min at 7° C. under an argon atmosphere. The reaction mixture wasextracted with ethyl acetate (2×50 mL) and washed with water (50 mL).The organic layer was dried over anhydrous Na₂SO₄, filtered andevaporated. The resulting free base residue (5, R₁=benzyl) as a viscousoil was used in the next reaction without any further purificationMS(ESI) 248(M+H)

Example 4. Synthesis of ONC202 (Compound (14))

To a solution of 2-chlorobenzylamino-2-imidazoline (150 mg, 0.72 mMol),methyl 1-benzyl 4-oxo-3-piperidine carboxylate (5, R₁=benzyl) (195 mg,0.79 mMol) in 1-butanol (2 mL) was added PPTS (10 mg) and the mixturewas stirred at room temperature for 48 h. After that the reactionmixture was refluxed at 125° C. to 130° C. for 2 h. The solvents wereremoved under vacuum, extracted with ethyl acetate (10 mL), and washedwith saturated sodium bicarbonate solution (2×10 mL) and water (10 mL).The organic layer was dried over anhydrous Na₂SO₄, filtered andevaporated. The crude free base was purified by RP HPLC (10%-40%acetonitrile/water) to give ONC202 TFA salt as a white solid (228 mg,50% yield) MS(ESI) 407 (M+H).

The same process was used starting with different benzylamines toprepare various analogs, e.g., ONC203, 204, 205, 206, 912, 210, 211,212, 213, 214, 217, 218, 219, 220, 221, 222, 223, 224, 225, and 226.

Example 5. Synthesis of ONC207 (Compound (19))

To a suspension of 60% sodium hydride (3.5 g, 88 mMol) in dry toluene(50 mL), dimethyl carbonate (4.32 g, 48.0 mMol) was added dropwise in0.5 h at room temperature under nitrogen. After addition of a few dropsof methanol, 1-tert-butoxycarbonyl-4-piperidone (4.8 g, 24 mMol)dissolved in dry toluene (20 mL) was added dropwise to the reactionmixture while stirring at 80° C. over 1 h. The reaction mixture wasstirred for 3 h at the same temperature and then cooled to 0° C. (icebath) and adjusted to pH 6-6.5 with acetic acid. The resulting coldmixture was diluted with water (10 mL) and adjusted to pH 8 with 5%sodium hydroxide solution. The toluene layer was separated and theaqueous layer was extracted with toluene (20 mL). The combined organiclayer was dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The compound was dried in vacuum to givemethyl-1-tert-butoxycarbonyl-4-oxo-3-piperidine carboxylate (5.0 g,80%). The compound obtained was carried to the next reaction without anyfurther purification.

2-methybenzylamino-2-imidazoline (190 mg, 1 mMol), methyl1-tert-butoxycarbonyl-4-oxo-3-piperidine carboxylate (315 mg, 1.1 mMol)in 1-butanol (2 mL) was added PPTS (10.0 mg) and the mixture was stirredat room temperature for 48 h. After that the reaction mixture wasrefluxed at 125° C. to 130° C. for 2 h. The solvents were removed undervacuum, extracted with ethyl acetate (10 mL), washed with saturatedsodium bicarbonate solution (2×10 mL) and water (10 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and evaporated. Thecrude free base was cleaved with 10% trifluoroacetic acid indichloromethane, purified by RP HPLC (10%-40% acetonitrile/water) togive ONC207 (262 mg, 50%) TFA salt as a white solid MS(ESI) 297 (M+H).

Example 6. Synthesis of ONC209 (Compound (21))

A mixture of ONC207 (100 mg, 0.2 mMol), phenylethyl bromide (55.0 mg,0.28 mMol) and potassium carbonate (150 mg, 1.0 mMol) inN,N-dimethylformamide (3 mL) was heated to 70° C. for 12 h. The solventswere removed under vacuum, extracted with ethyl acetate (10 mL), andwashed with water (5 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and evaporated. The crude free base was purified by RPHPLC (10%-40% acetonitrile/water) to give ONC209 (62 mg, 50%) TFA saltas a white solid MS(ESI) 401 (M+H).

The same process was used starting with different halides to give ONC215and 214. Compounds 227, 228, 229, 230, 231, 232, 233, 234, 235, and 236were prepared using an analogous process from Examples 1 and 5 startingwith different benzylamines. Then treating the intermediate compoundwhere R₁ is H with different halides as above.

Compound ONC216 was prepared from ONC215 by treatment with TFA.

Compound (72) was prepared by reacting the precursor NH compoundprepared in analogy to Example 5 and treating it with styrene oxide.

Example 7. Synthesis of ONC208 (Compound (20))

To a solution of 2-methylbenzylamino-2-imidazoline (190.0 mg, 1.0 mmol),methyl 1-methyl 4-oxo-3-piperidine carboxylate (185.0 mg, 1.0 mMol) in1-butanol (2.0 mL) was added PPTS (10.0 mg) and the mixture was stirredat room temperature for 48 h. After that the reaction mixture wasrefluxed at 125° C. to 130° C. for 2 h. The solvents were removed undervacuum, extracted with ethyl acetate (10 mL), washed with saturatedsodium bicarbonate solution (2×10 mL) and water (10 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and evaporated. Thecrude free base was purified by HPLC 10%-40% acetonitrile and water togive ONC208 (270.0 mg, 50%) TFA salt as a white solid MS(ESI) 311 (M+H).

Example 8. Synthesis of ONC201 (Compound (1))

To a stirred 800 mL saturated NaHCO₃ in a 2 L round bottom flask,compound (3) (239.7 g, 0.845 mol, 1.6 equiv) was added in portions.n-Butanol (500 mL) was added to the resulting mixture, which was stirredfor 30 min and then transferred to a separating funnel. The organicphase, containing compound (4), was separated and transferred to a 2 Lthree-neck round bottom flask equipped with mechanical stirring, N₂inlet, a thermocouple, a condenser and a Dean-Stark trap. To thecontents of the flask, Compound (5) (100 g, 0.528 mol, 1 equiv) andpyridinium p-toluenesulfonate (PPTS) (6.63 gm 0.026 mol, 5 mol %) wereadded. The resulting mixture was heated to reflux for 6 hours. Water inthe reaction mixture was separated into the Dean-Stark trap asnecessary. Refluxing temperature increased from 93° C. to 118° C.Reaction progress was monitored by HPLC. When the peak area of compound(1) on HPLC remained constant with the reaction time, the reaction wasstopped.

Example 9. Synthesis of Di-Salt of ONC201 (Compound (1)*2 HCl)

Without isolation of the compound (1), the reaction mixture from Example8 was washed with water (500 mL) and diluted with methyl tert-butylether (MTBE) (800 mL). The organic phase was washed with water (500mL×2) and transferred to a 3 L three-neck round bottom flask equippedwith mechanical stirring, N₂ inlet, a thermocouple, a condenser and aDean-Stark trap. While agitating the reaction mixture, 1 N HCl indioxane-MTBE solution was added dropwise (4 N HCl in dioxane: 300 mL,1.2 mol, 2.27 equiv; MTBE: 1200 mL) until no more solid precipitated outof the reaction mixture upon addition of HCl. The reaction mixture washeated to reflux at 60-65° C. for 2 hours. Water was separated into theDean-Stark trap as necessary. Upon cooling to room temperature, thesolid precipitate was filtered through a sintered glass funnel andwashed with n-butanol-MTBE (1:2, 600 mL) and MTBE (600 mL) respectively.The solid was dried in a vacuum oven at 65° C. overnight (16 hours) toafford 200 g yellow solid.

To a 2 L three-neck round bottom flask equipped with mechanicalstirring, N₂ inlet, a thermocouple and a condenser, the above solid (200g) was added, followed by ethanol (1000 mL). The mixture was heated toreflux at 78° C. for 2 hours. Upon cooling to room temperature, thesolid was filtered through a sintered glass funnel and washed withethanol (200 mL×3). The wet solid was dried in the vacuum oven at 85° C.for 3 days until the residual solvent met specification. 120 g ofcompound (2) was obtained as a white solid in a yield of 49%, with HPLCpurity 99.7%.

Example 10. Activity of Imipridones

A number of imipridones were prepared based on the syntheses above.Viability of human cancer cells was measured at 72 hours post-treatmentwith each compound. The change in potency (relative to ONC201) wasdetermined and shown in Table 3.

TABLE 3 RELATIVE POTENCY OF ONC201 ANALOGS Relative No. Identifier R₁ R2Potency*  1 ONC201 CH₂Ph CH₂—((2-CH₃)-Ph) N/A 14 ONC202 CH₂PhCH₂(2-Cl-Ph) B 15 ONC203 CH₂Ph CH₂—(2-thienyl) C 16 ONC204 CH₂PhCH₂CH₂Ph C 17 ONC205 CH₂Ph CH₂CH₂(4-N-benzyl-piperazine) C 18 ONC206CH₂Ph CH₂—(2,4-di F—Ph) A 19 ONC207 H CH₂—((2-CH₃)—Ph) B 20 ONC208 CH₃CH₂—((2-CH₃)—Ph) B 21 ONC209 CH₂CH₂Ph CH₂—((2-CH₃)—Ph) B 32 ONC215(CH₂)₃—NH—BOC CH₂—((2-CH₃)—Ph) B 33 ONC216 (CH₂)₃—NH₂ CH₂—((2-CH₃)—Ph) B41 ONC210 CH₂Ph CH₂—(3,5-di F—Ph) B 51 ONC211 CH₂Ph CH₂—(3,4-di Cl—Ph) B52 ONC212 CH₂Ph CH₂—(4-CF₃—Ph) A 53 ONC213 CH₂Ph CH₂—(3,4-di F—Ph) A 54ONC214 CD₂C₆D₅ CH₂—((2-CH₃)—Ph) B 43 ONC217 CH₂Ph CH₂(2-F—Ph) C 55ONC218 CH₂Ph CH₂(2-CH₃, 4-F—Ph) A 56 ONC219 CH₂Ph CH₂—(2,4-di Cl—Ph) A57 ONC220 CH₂Ph CH₂—((4-OCH₃)—Ph) A 35 ONC222 CH₂PhCH₂—(3-isoxazolidinyl) C 36 ONC224 CH₂Ph CH₂CH₂—(4-morpholinyl) A 38ONC221 H CH₂—(4-CF₃—Ph) A 72 ONC225 CH₂Ph CH₂—(2-F, 4-CF₃—Ph) A 37ONC223 CH₂Ph CH₂—(4-CH₃—Ph) A 34 ONC226 CH₂Ph CH₂—(3-pyridinyl) A 77ONC231 CH₂—3-pyridyl CH₂—(4-CF₃—Ph) A 78 ONC232 CH₂—4-methyl-2-thiazolylCH₂—(4-CF₃—Ph) B 79 ONC233 CH₂—2-pyrazinyl CH₂—(4-CF₃—Ph) B 81 ONC234CH₂—(3,4-di Cl—Ph) CH₂—(4-CF₃—Ph) A 83 ONC236 CH₂—3-thienylCH₂—(4-CF₃—Ph) A 84 ONC237 CH₂CH(OH)Ph CH₂—(4-CF₃—Ph) C 73 ONC227CH₂—(4-CF₃—Ph) CH₂—(4-CF₃—Ph) B 74 ONC228 CH₂—(4-F—Ph) CH₂—(4-CF₃—Ph) A75 ONC229 CH₂—(4-OCH₃—Ph) CH₂—(4-CF₃—Ph) B 76 ONC230 4-F—Ph-4-oxobutylCH₂—(4-CF₃—Ph) A *Relative to the potency of ONC201; A Indicates apotency increase of >2-fold of ONC201; B Indicates potency that iswithin 2-fold of ONC201; and C Indicates a potency decrease of >2-foldof ONC201.

The IC₅₀ of ONC201 and ONC212 (5 nM-5 μM, 72 h) upon treatment ofseveral acute myeloid leukemia (AML) cell lines (n=3) were determinedand shown in Table 4.

TABLE 4 AML ONC201 ONC212 cell line IC₅₀ (μM) IC₅₀ (μM) MV411 3.25 0.01HL60 >5 0.21 MOLM14 3.92 0.01

Cell viability of MV411 AML cells treated with ONC212 and cytarabine (5nM-5 μM, 24 h) (n=3) was measured (FIG. 14A). Furthermore, cellviability MOLM14, MV411 AML cells, MRC5 lung fibroblasts and Hs27a bonemarrow cells treated with ONC212 (5 nM-5 μM, 72 h) (n=3) was measured(FIG. 14B). Cell viability of MOLM14 and MV411 AML cells treated withONC212 (250 nM) for 4, 8, 24, 48, 72 and 96 h was measured. ONC212medium was replaced by fresh medium at these time points and cellviability was determine at 96 h for all samples. (n=2) (FIG. 14C).

In addition, a single dose of ONC212 by oral or intraperitonealadministration to human colon cancer xenograft-bearing mice resulted insignificant reduction of tumor volume compared to vehicle-treatedcontrol cohorts (FIG. 10). ONC212 has a wide therapeutic window, as itis well tolerated at doses at least up to 225 mg/kg in mice.

Furthermore, ONC212 demonstrated efficacy in ONC201-resistant AMLxenograft model (FIG. 15). MV411 AML cells (5×10⁶) were subcutaneouslyimplanted in the flanks of athymic nude. ONC212 and ONC201 wereadministered orally (PO) as indicated. Tumor volume (A and B) and bodyweight (C) (n=10) was measured on indicated days. * represents p<0.05relative to vehicle.

ONC212 efficacy in AML was evaluated in vitro and was up to 400 foldmore potent compared to ONC201 (Table 4). ONC212 was also efficacious inAML cells resistant to standard of care cytarabine (FIG. 14A). Despiterobust improvement in efficacy ONC212 maintains a wide therapeuticwindow in vitro and is non-toxic to normal cells at efficaciousconcentrations (FIG. 14B). An 8 hr exposure of ONC212 at 250 nM wassufficient to cause robust reduction in cell viability in MOLM14 andMV411 AML cells (FIG. 14C). At least 24-48 h exposure was required withONC201 for efficacy.

ONC212 efficacy was determined in a leukemia xenograft model with MV411AML cells resistant to standard-of-care cytarabine (FIG. 15). ONC212 50mg/kg significantly reduced leukemia xenograft tumor growth with oralweekly administration while ONC201 was not efficacious in this model atsimilar doses (FIG. 15A). Interesting, biweekly ONC212 dosing with 25mg/kg and weekly/biweekly dosing with 5 mg/kg was not efficacious (FIG.15B). None of these ONC212 administration regimens were associated withbody weight loss (FIG. 15C) or gross observations.

ONC212 25 mg/kg represents NOAEL in mouse and rat non-GLP oral singledose studies which is also the efficacious dose in mouse xenograftstudies. ONC212 is approximately 10 fold more toxic compared to ONC201(NOAEL 225 mg/kg in rat non-GLP oral single dose study).

ONC206 demonstrated efficacy in a Ewing's sarcoma xenograft model.MHH-ES-1 Ewing's sarcoma cells (5×10⁶) were subcutaneously implanted inthe flanks of athymic nude mice. ONC206 (PO) and methotrexate (IV) wereadministered on day 1 and day 13 as indicated. Tumor volume (FIG. 16A)and body weight (FIG. 16B) (n=4) was measured on indicated days.

In addition, the IC₅₀ of ONC201 and ONC206 (5 nM-5 μM, 72 h) upontreatment of several cell lines (n=3) were determined and shown below inTable 5.

TABLE 5 Cell ONC201 ONC206 line IC₅₀ (μM) IC₅₀ (μM) MV411 (AML) 3.25 0.2K562 (CML) >5 0.22 MOLM14 (AML) 3.92 0.27 MHH-ES-1 5.65 0.61 (Ewing'ssarcoma) HFF (Normal Fibroblast) >5

ONC206 showed up to 20 fold improvement compared to ONC201 in in vitropotency with no in vitro toxicity to normal cells at therapeutic doses(Table 5). With ONC206, only 2-fold increased toxicity (NOAEL 125 mg/kg)was noted overall relative to ONC201 (NOAEL 225 mg/kg) in rat non-GLPoral single dose study. In vivo efficacy in Ewing's sarcoma model withno toxicity (FIG. 16). ONC206 efficacy was comparable to chemotherapymethotrexate, but chemotherapy was associated with body weight loss.

In vitro profiling of GPCR activity using a hetereologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC213 selectively targets DRD2/3 and GPR132/91. Dual targeting ofDRD2/3 and GPR132/91 represents a novel strategy for anti-cancerefficacy without toxicity. ONC213 is a DRD2/3 inhibitor and a GPR132/91agonist. DRD2/3 potency of ONC213 is more than ONC201 but less thanONC206. GPR132 potency of ONC213 is less than ONC212. Specifically,ONC213 demonstrated in vitro anti-cancer potency in HCT116/RPMI8226cancer cells similar to ONC212, but in vitro toxicity to normal cellswas reduced compared to ONC212 (FIG. 17). The safety profile of ONC213confirmed in mouse MTD study with NOAEL 75 mg/kg three times that ofONC212 (25 mg/kg). The GPR91 agonist activity of ONC213 provides anopportunity for immunology, immune-oncology and hematopoieticapplications (Nature Immunology 9:1261 (2008); J Leukoc Biol. 85(5):837(2009)).

In vitro profiling of GPCR activity using a hetereologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC237 selectively targets DRD5 and GPR132. ONC237 is a GPR132 agonistand DRD5 antagonist and has reduced anticancer efficacy (IC₅₀ 31.2 μM)compared to ONC201. This data show that combining GPR132 agonistactivity with DRD5 (D1-like dopamine receptor) antagonist activityresults in poor anti-cancer effects compared to ONC213 which combinesGPR132 agonist and DRD2/3 antagonist activity.

In vitro profiling of GPCR activity using a hetereologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC236 is a highly selective GPR132 agonist. ONC236 has anticancerefficacy (IC₅₀ 88 nM) comparable to ONC212 (10 nM) better thanONC206/ONC201, completeness of response is better than ONC201 but notONC212 in HCT116 cells.

In vitro profiling of GPCR activity using a hetereologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC234 is a broad spectrum and potent GPCR targeting small molecule.ONC234 hits several GPCRs including antagonist activity for adrenergic,histamine, serotonin, CHRM, CCR, DRD2/5 receptors, as well as CXCR7agonist activity. ONC236 has anticancer efficacy (IC₅₀ 234 nM) similarto ONC206, completeness of response same as ONC212, and better thanONC201 in HCT116 cells.

Example 11. GPCR Antagonism of ONC201

ONC201 was evaluated in a whole cell, functional assay of β-Arrestin Gprotein-coupled receptor (GPCR) activity that directly measures dopaminereceptor activity by detecting the interaction of β-Arrestin with theactivated GPCR that serves as a reporter. For each dopamine receptor(DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5), cell lines overexpressingreporter constructs were expanded from freezer stocks. Cells were seededin a total volume of 20 μL into white walled, 384-well microplates andincubated at 37° C. prior to testing, with antagonist followed byagonist challenge at the EC₈₀ concentration. Intermediate dilution ofsample stocks was performed to generate 5× sample in assay buffer. 3.5μL of 5× sample was added to cells and incubated at 37° C. or roomtemperature for 30 minutes. Vehicle concentration was 1%. 5 μL of 6×EC₈₀agonist in assay buffer was added to cells and incubated at 37° C. orroom temperature for 90 or 180 minutes prior to assay readout. %Antagonism was calculated using the following formula %:Antagonism=100%×(1−(mean RLU of test sample−mean RLU of vehiclecontrol)/(mean RLU of EC₈₀ control−mean RLU of vehicle control).

Example 12: Selective Antagonism of DRD2 by ONC201

ONC201 is a first-in-class small molecule discovered in a phenotypicscreen for p53-independent inducers of tumor selective proapoptoticpathways. Oral ONC201 is being evaluated as a new therapeutic agent infive early phase clinical trials for select advanced cancers based onpronounced efficacy in aggressive and refractory tumors and excellentsafety.

In this Example, the prediction and validation of selective directmolecular interactions between ONC201 and specific dopamine receptorfamily members are reported. Experimental GPCR profiling indicated thatONC201 selectively antagonizes the D2-like, but not D1-like, dopaminereceptor subfamily. Reporter assays in a heterologous expression systemrevealed that ONC201 selectively antagonizes both short and longisoforms of DRD2 and DRD3, with weaker potency for DRD4 and noantagonism of DRD1 or DRD5. Increased secretion of prolactin is aclinical hallmark of DRD2 antagonism by several psychiatric medicationsthat potently target this receptor. ELISA measurements in peripheralblood of patients treated with ONC201 in the first-in-human trial withadvanced solid tumors determined that 10/11 patients evaluated exhibitedinduction of prolactin (mean of 2-fold).

Using the TCGA database, the D2-like dopamine receptor subfamily,particularly DRD2, was found to be prevalent and selectivelyoverexpressed in several malignancies. Preclinical reports show thatDRD2 inhibition imparts antitumor efficacy, without killing normalcells, via induction of ATF4/CHOP and inhibition of Akt and ERKsignaling that are all attributes of ONC201.

Methods

ONC201 dihydrochloride was obtained from Oncoceutics. Kinase inhibitionassays for the kinome were performed as described (see Anastassiadis etal., Nat Biotech 29:1039 (2011)). GPCR arrestin recruitment and cAMPmodulation reporter assays were performed as described (see McGuinnesset al., Journal of Biomolecular Screening 14:49 (2009)). PathHunter™(DiscoveRx) β-arrestin cells expressing one of several GPCR targets wereplated onto 384-well white solid bottom assay plates (Corning 3570) at5000 cells per well in a 20 μL volume in an appropriate cell platingreagent. Cells were incubated at 37° C., 5% CO₂ for 18-24 h. Sampleswere prepared in buffer containing 0.05% fatty-acid free BSA (Sigma).For agonist mode tests, samples (5 μL) were added to pre-plated cellsand incubated for 90 minutes at 37° C., 5% CO₂. For antagonist modetests, samples (5 μL) were added to pre-plated cells and incubated for30 minutes at 37° C., 5% CO₂ followed by addition of EC₈₀ agonist (5 μL)for 90 minutes at 37° C., 5% CO₂. For Schild analysis, samples (5 μL)were added to pre-plated cells and incubated for 30 minutes at 37° C.,5% CO₂ followed by addition of serially diluted agonist (5 μL) for 90minutes at 37° C., 5% CO₂. Control wells defining the maximal andminimal response for each assay mode were tested in parallel. Arrestinrecruitment was measured by addition of 15 μL PathHunter Detectionreagent and incubated for 1-2 h at room temperature and read on a PerkinElmer Envision Plate Reader. For agonist and antagonist tests, data wasnormalized for percent efficacy using the appropriate controls andfitted to a sigmoidal dose-response (variable slope),Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogEC₅₀−X)*HillSlope)), where X is the log concentration of compound. ForSchild analysis, data was normalized for percent efficacy using theappropriate controls and fitted to a Gaddum/Schild EC₅₀ shift usingglobal fitting, where Y=Bottom+(Top-Bottom)/(1+10{circumflex over( )}((Log EC−X)*HillSlope)), Antag=l+(B/(10{circumflex over( )}(10*pA2))){circumflex over ( )}SchildSlope and LogEC=Log(EC₅₀*Antag). EC₅₀/IC₅₀ analysis was performed in CBIS dataanalysis suite (Cheminnovation) and Schild analysis performed inGraphPad Prism 6.0.5.

Results

ONC201 is a small molecule in phase II clinical trials for selectadvanced cancers. It was discovered in a phenotypic screen forp53-independent inducers of the pro-apoptotic TRAIL pathway. Althoughthe contribution of ONC201-induced ATF4/CHOP upregulation andinactivation of Akt/ERK signaling (Allen et al., Science translationalmedicine 5, 171ra117-171ra117 (2013)) to its anti-cancer activity hasbeen characterized, its molecular binding target had remained elusive.

In vitro profiling of GPCR activity using a hetereologous reporter assayfor arrestin recruitment, a hallmark of GPCR activation, indicated thatONC201 selectively antagonizes the D2-like (DRD2/3/4), but not D1-like(DRD1/5), dopamine receptor subfamily (FIG. 1). Antagonism ofadrenoceptor alpha receptors or other GPCRs was not observed under theevaluated conditions. Among the DRD2 family, ONC201 antagonized bothshort and long isoforms of DRD2 and DRD3, with weaker potency for DRD4.Further characterization of ONC201-mediated antagonism of arrestinrecruitment to DRD2L was assessed by a Gaddum/Schild EC₅₀ shiftanalysis, which determined a dissociation constant of 2.9 μM for ONC201that is equivalent to its effective dose in many human cancer cells.Confirmatory results were obtained for cAMP modulation in response toONC201, which is another measure of DRD2L activation. The ability ofdopamine to reverse the dose-dependent antagonism of up to 100 μM ONC201suggests direct, competitive antagonism of DRD2L. In agreement with theONC201 specificity predicted by BANDIT, no significant interactions wereidentified between ONC201 and nuclear hormone receptors, the kinome, orother drug targets of FDA-approved cancer therapies. Interestingly, abiologically inactive constitutional isomer of ONC201 (Wagner et al.,Oncotarget 5:12728 (2014)) did not inhibit DRD2L, suggesting thatantagonism of this receptor could be linked to its biological activity.In summary, these studies establish that ONC201 selectively antagonizesthe D2-like dopamine receptor subfamily, which appears to be a promisingtherapeutic target in oncology, and ONC201 is the first compound toexploit this treatment paradigm in several ongoing Phase II clinicalstudies.

Example 13: Preclinical Data in H3 K27M Adult and Pediatric Glioma

The discovery of H3 K27M as an oncogenic mutation occurred in thecontext of midline gliomas that involve the thalamus, pons, or spinalcord. H3 K27M refers to a specific mutation in histone H3 proteins. Dueto the location of these tumors, areas of the brain involved in criticalphysiological functions, these tumors have historically been inoperable(especially in the brain stem where the pons is located). This meansthat until recently, midline gliomas such as diffuse intrinsic pontineglioma (DIPG) were diagnosed solely on a radiographic basis. Recentadvances in neurosurgical techniques and increased parental consent topost-mortem tumor tissue retrieval led to the availability of sufficientbiospecimens that enabled systematic genomic evaluations of DIPG andother midline gliomas. Gliomas in the midline of the brain belong to themost aggressive types of primary malignant brain cancers. The diseasearises from glial cells, which are cells that form the tissue thatsurrounds and protects other nerve cells found within the brain andspinal cord.

Standard therapy for midline gliomas involves neurosurgery, whenfeasible, followed by fractionated external beam radiotherapy. Due tolocation in the brain, aggressiveness and low survival time, gliomas inthe midline of the brain are considered as part of the most lethal formsof cancer.

There is evidence that H3 K27M predominantly occurs in midline gliomasand in younger patients: ˜75% of thalamic brain tumors, ˜54% ofbrainstem tumors and 55% of spinal cord tumors; 24% of pediatric gliomasand 8% of adult gliomas. The H3 K27M mutation occurs in a uniquespatiotemporal pattern, with midline gliomas involving the pons (i.e.DIPG) tending to occur in pediatric patients (<18 years of age) whilemidline gliomas involving the thalamus and spinal cord tending to occurin young adult patients.

The presence of the H3 K27M mutation in midline gliomas is generallythought to confer a worse clinical prognosis. This understanding wasincorporated into the World Health Organization 2016 classification ofcentral nervous system tumors that now defines diffuse midline gliomaswith the H3 K27M mutation as a new distinct disease entity. This diseaseis defined as grade IV regardless of histopathological features due tothe widely recognized dismal prognosis of brain tumors with thismutation.

Most of the prognostic literature for H3 K27M is derived from DIPG thatexhibits a 70-85% prevalence of this mutation. It is clear than thepresence of the H3 K27M mutation in tumors of the pons confers a muchshorter overall survival relative to the minority of patients who do nothave this mutation. For the smaller number of pediatric patients withgliomas outside of the pons, the literature is consistent that thosewith the H3 K27M mutation have a poorer prognosis. The field looks toDIPG as the most robust body of clinical experience with H3 K27M gliomasbased on high prevalence of the mutation in that disease. Decades ofDIPG clinical trials have failed to improve outcomes andstandard-of-care, a 6-week course of radiation, remains associated witha 9-11-month overall survival. Historically, therapeutic clinical trialsin DIPG focused on the evaluation of therapies that were proveneffective in adult high-grade gliomas. The recent molecular profilingand emerging preclinical models of H3 K27M midline gliomas have shownthat these tumors exhibit vastly different biology and therapeuticsensitivity relative to other adult gliomas, such as glioblastoma.

H3 K27M-mutant gliomas occur at a lower rate in adults compared topediatric patients. The literature is relatively congruous withpediatric findings and overall seems to confirm the dismal effect of H3K27M mutations in brain tumors for adults, especially in brainstemgliomas. Overall survival of adult patients with H3 K27M midline gliomasis approximately 16 months with studies indicating that H3 K27Mmutations in brainstem locations are associated with significantlyshorter survival times.

One of the features in the selection process that identified ONC201 asan anticancer agent was the compound's ability to penetrate theblood-brain barrier to address tumors residing in the CNS, unlike manyavailable therapies. Ensuing animal studies revealed that ONC201 rapidlytraverses the blood-brain barrier, achieves 5-fold higher concentrationsin the brain relative to plasma and induces downstream signaling (TRAILinduction) in the brain.

The compound is highly bioactive in the brain, shows no evidence ofneurotoxicity, and is potently cytotoxic to high grade glioma tumors invitro, ex vivo, and in vivo. ONC201 has p53-independent activity againsthigh grade glioma cell lines including those with resistance toradiotherapy. In addition to cell lines, ONC201 exerts potent anticanceractivity in primary high grade glioma samples resistant to temozolomide.

In vivo, ONC201 shrinks temozolomide-resistant high-grade gliomaxenografts and prolongs the survival of mice with orthotopic xenograftsas a monoagent and in combination with bevacizumab. Compelling monoagentefficacy of ONC201 has also been observed in radio- and chemo-resistanthigh-grade glioma cell lines and in 3D neurosphere cultures of newlydiagnosed and recurrent patient samples.

DRD2 is overexpressed in high grade glioma. In studies utilizing sixhuman GBM cell lines from the NCI60 panel of cell lines, expressionlevels of DRD2 correlated with the responsiveness of the cells to ONC201(FIG. 3). Interestingly, expression of DRD5, a D1-like dopamine receptorthat counteracts DRD2 signaling, was significantly inversely correlatedwith ONC201 potency in the NCI60 and GDSC datasets (P<0.05) (FIG. 3).

In publically available ChIP-Seq databases, H3 and components of thePRC2 methyl transferase complex, which is inhibited by the K27Mmutation, were found to each mark both the DRD2 and DRD5 gene in DIPGand isogenic models. While the precise epigenetic mechanisms regulatingthe balance of the DRD2:DRD5 expression is an active area ofinvestigation, H3 K27M gliomas was hypothesized to foster a chromatinlandscape that leads to high DRD2 expression and suppression of DRD5expression, which in turn may make these tumor cells more sensitive toONC201. ONC201 was tested against a panel of patient-derived gliomatumorsphere cultures grown in serum-free neural stem cell media.Patient-derived lines included five histone H3 K27M mutant DIPG (twoHIST1H3B and three H3F3A mutant), two H3F3A G34 mutant pediatricglioblastoma (one G34V, one G34R), and 7 H3 wild-type (3 pediatric, 4adult) glioblastoma cell lines. ONC201 was more potently cytotoxic tohistone H3 K27M mutant (median IC₅₀-0.6 μM, n=5 lines) compared tohistone H3 wild-type glioma lines (median IC₅₀˜1.5 μM, n=7 lines;p<0.01).

In addition, the expression of DRD2 and DRD5 was analyzed in untreatedpatient glioma samples. RNASeq was conducted on patient biopsies from H3K27M mutant glioma (n=8), wild-type pediatric (n=3) and adult glioma(n=25), H3 G34R mutant glioma (n=3). DRD2 expression was significantlyincreased in histone H3 K27M mutant glioma tumors compared to adult andpediatric H3 wild-type tumors (p=0.02). In contrast, DRD5 expression inall glioma tumors tested were low, however DRD5 expression in histone H3K27M mutant glioma tumors showed a trend towards lower expression thanwild-type glioma. Therefore, DRD2 and DRD5 expression profiles of H3K27M mutant patient gliomas appear consistent with an expressionsignature in preclinical models that predicts ONC201 sensitivity.

Cancer stem cells have been shown to express relatively high levels ofDRD2 compared to the bulk population, and ONC201 effectively depletescancer stem cells in numerous malignancies. This effect may contributeto the prolongation of survival in patients in a Phase II GBM study ofONC201 despite the fact that many patients received limited exposure todrug (1 or 2 doses).

NK cells are known to express DRD2 and ONC201 has been reported toincrease the pool of circulating and intratumoral NK cells. Even moreimportant is activation of NK cell function documented in vivo as wellas in patient samples. This significant immune-stimulatory effect likelycontributes to the antitumor activity of the compound and is consistentwith the response kinetics, i.e. prolonged and late responses, seen inan ongoing clinical trial. The GBM tumor microenvironment has beendescribed as profoundly immune-suppressed and several modalities thatstimulate immune function have been shown to affect GBM tumor cellgrowth.

Example 14: ONC201 Treatment in a 22 Year Old Female with Recurrent H3K27M Mutant Glioblastoma

Histone H3 K27M mutations distinguish a subgroup of midline gliomas inchildren and young adults with devastating prognosis for which there areno effective medical therapies. The first H3 K27M glioma patient toreceive ONC201 was a 22-year-old female with multi-focal disease thatincluded her thalamus who was treated as part of a Phase II recurrentglioblastoma trial in adults (NCT02525692). She had recurrentglioblastoma (unmethylated MGMT, H3.3 K27M mutant) and was treated with625 mg of ONC201 once every three weeks. She had previously progressedfollowing prior surgery, radiation, and temozolomide.

After initiating ONC201 therapy, this subject showed a durable objectiveresponse with complete regression of thalamic lesions (FIGS. 13A and18A). Overall tumor size regressed by 96% after 18 doses (FIGS. 13B and18B). She no longer takes anti-seizure medication on a regular basis, asshe did prior to initiating ONC201. This response remains durable andshe continues therapy for >1.5 years with no drug-related adverse eventsreported. In addition to cytotoxic effects in tumor cells, DRD2antagonism can induce the activation of NK and other immune cells.Immune induction correlated with tumor shrinkage (FIG. 19). Immuneeffector levels in the serum was found to correlate with the kinetics ofthe objective response, which is consistent with a delayed durableresponse.

Example 15: ONC201 Treatment in a 74 Year Old Female with Recurrent H3K27M Mutant Glioblastoma

This Example provides a case study of a 74-year-old female H3 K27M(unknown MGMT status) glioma patient who also participated in the PhaseII recurrent glioblastoma trial with ONC201 referenced in the previousExample. She also had multi-focal disease and progressed followingfirst-line surgery, radiation, and temozolomide. She also progressedfollowing subsequent second-line therapy with CCNU. Prior to ONC201treatment, this subject had three lesions. Her first on-treatmentevaluation at 8-weeks following initiation of ONC201 therapy revealed acomplete disappearance of malignant lesions (FIG. 20). She alsotolerates the therapy well and remains on study after >10 weeks.

Example 16: ONC201 Treatment in a 10 Year Old Girl with H3 K27M MutantDIPG

In this Example, a 10 year old girl with H3.3 K27M mutant diffuseintrinsic pontine glioma (DIPG) was treated with 500 mg of ONC201 onceweekly. The subject suffered from left facial palsy and decreasedhearing in the left ear. Prior therapy included a 6 week course ofradiation. After 16 weeks of ONC201 therapy, radiographic evaluation ofher tumor revealed a significant regression and decreased enhancement ofher exophytic cerebral tumor (FIG. 21). She also experiencednear-complete resolution of her facial palsy that was associated with acranial nerve palsy due to her tumor's location. She also tolerates thetherapy well and continues on trial.

Example 17: ONC201 Treatment in a 3 Year Old Girl with H3 K27M MutantDIPG

In this Example, a 3 year old girl with H3.3 K27M mutant diffuseintrinsic pontine glioma (DIPG) was treated with 125 mg of ONC201 onceweekly. The subject suffered from right sixth nerve palsy and left armand hand weakness. Prior therapy included a 6 week course of radiation.After 6 weeks of ONC201 therapy, radiographic evaluation of her tumorrevealed a stable tumor lesion (FIG. 22). She also experienced acomplete resolution of her inability to use her left arm and hand thatwas associated with a cranial nerve palsy due to her tumor's location.She is fully ambulatory and also tolerates the therapy well andcontinues on trial.

High grade gliomas with the H3 K27M mutation have significantly inferiorclinical outcomes relative to patients who do not have the mutation. Inone study of thirty-nine pediatric diffuse intrinsic pontine gliomas,DIPG patients carrying the K27M-H3.3 mutation (n=30) have worse overallsurvival compared to patients wild-type (n=9) for this histone(Khoung-Quang et al., Acta Neuropathol (2012) 124:439). Notably, alllong-term survivors were H3.3 wild type. Surprisingly, ONC201 treatmentconfers superior progression-free survival (PFS) in H3 K27M patients.Progression-free survival (PFS) with ONC201 treatment was determined forfifty patients with recurrent high grade glioma present at baseline byMRI before initiating ONC201 therapy (FIG. 23). The cohort is dividedinto two groups: one with known H3 K27M mutation (n=15) and the otherwith wild-type or unknown H3 status (n=35). Notably, all long-termprogression-free patients treated with ONC201 were H3 K27M patients.

Example 18: Clinical Evaluation of the Imipridone ONC201 in RecurrentGlioblastoma: Predictive and Pharmacodynamic Biomarker Analyses

The imipridone ONC201 is the first selective antagonist of the Gprotein-coupled receptor DRD2 for clinical oncology. ONC201 inducesp53-independent apoptosis in newly diagnosed and recurrent glioblastomain vitro, ex vivo, and in vivo. A Phase II clinical trial was performedthat enrolled an initial cohort of 17 patients with recurrent,bevacizumab-naïve, IDH1/2 WT glioblastoma who received 625 mg ONC201every three weeks. One patient continues to have a durable objectiveresponse that has deepened over time, exhibiting an 92% regression by92% after 15 months of therapy. Another patient remains disease-free 14months after enrolling on this trial following a re-resection. Median OSwas 41.6 weeks with an OS12 of 35%. No drug-related SAEs or treatmentdiscontinuation due to toxicity occurred. Plasma PK at 2 hours post-dosewas 2.6 μg/mL and serum prolactin induction was observed as a surrogatemarker of target engagement. In addition to cytotoxic effects in tumorcells, DRD2 antagonism can induce the activation of NK and other immunecells. Immune effector levels in the serum correlated with the kineticsof the objective response. Preclinical studies have identified aDRD2⁺DRD5⁻ tumor biomarker signature that is predictive of innatesensitivity to ONC201. Among the 15 available archival tumor tissuespecimens, all had expression of DRD2 and 8/17 patients had lowexpression of DRD5. Patients with PFS>5 month had no detectableexpression of DRD5 unlike those with PFS<5 months. In addition, 4/8DRD2⁺DRD5⁻ and 0/7 DRD2⁺DRD5⁺ patients still alive with a medianfollow-up of 47.4 weeks. In summary, ONC201 is a well tolerated therapywith potential anti-glioblastoma activity that may be associated with apredictive biomarker signature and immune activation.

Example 19: ONC201 is Active in Glioblastoma with DRD2 PathwayDysregulation

ONC201, an imipridone that is a selective antagonist of the Gprotein-coupled receptors dopamine receptor D2 (DRD2) and D3 (DRD3), hasexhibited tumor shrinkage and an exceptional safety profile in a phaseII recurrent glioblastoma clinical trial. In vitro and in vivo studieshave demonstrated single agent ONC201 efficacy in glioblastoma models(Allen et al., Science translational medicine 5, 171ra117-171ra117(2013)). In vitro efficacy profiling of ONC201 in the Genomic of DrugSensitivity in Cancer (GDSC) collection of cell lines confirmed broadanti-cancer efficacy with high sensitivity in human brain cancer celllines (FIG. 2). DRD2 is overexpressed in glioblastoma and DRD2antagonism induces tumor cell apoptosis via the same signaling pathwaysthat respond to ONC201. Investigation of The Cancer Genome Atlas (TCGA)revealed that DRD2 is highly expressed in glioblastoma relative to otherdopamine receptor family members and that genetic aberrations were rare.High expression of DRD2 occurred in primary, rather than secondary,glioblastoma and was associated with a poor prognosis.Immunohistochemistry analyses of tissue microarrays revealed DRD2overexpression in glioblastoma relative to normal brain. A linearcorrelation between DRD2 mRNA and ONC201 GI₅₀ was observed amongglioblastoma cell lines in the NCI60 panel. A significant induction ofserum prolactin, a surrogate biomarker of target engagement, wasdetected in ONC201-treated glioblastoma patients. Interestingly,expression of DRD5, a D1-like dopamine receptor that counteracts DRD2signaling, was significantly inversely correlated with ONC201 potency inthe NCI60 and GDSC datasets (P<0.05). A missense DRD5 mutation was alsoidentified in cancer cells with acquired resistance to ONC201.Resistance could be recapitulated with overexpression of the mutant DRD5gene or, to a lesser extent, with the wild-type gene. In conclusion, theDRD2 pathway is a therapeutic target that is dysregulated inglioblastoma and contains biomarkers of tumor sensitivity to ONC201.

Example 20: Differentiated Pharmacology of the Imipridone ONC201, theFirst Selective DRD2/3 Antagonist in Clinical Neuro-Oncology

ONC201, founding member of the imipridone class of compounds, hasdemonstrated evidence of tumor shrinkage along with exceptional safetyin recurrent glioblastoma patients. In this Example, a previouslyunknown binding target of ONC201 was identified and characterized.BANDIT—a machine learning-based drug target identificationplatform—predicted that ONC201 would bind with high selectivity to theG-protein coupled receptors (GPCRs) dopamine receptor D2 (DRD2) and D3(DRD3). DRD2 is overexpressed in glioblastoma, controls pro-survivalmechanisms, and its antagonism causes pro-apoptotic effects in malignantcells. PATHHUNTER® β-arrestin and cAMP assays determined that ONC201selectively antagonizes DRD2 and DRD3. Consistent with BANDIT and incontrast to DRD2 blocking antipsychotics, ONC201 did not antagonizeother dopamine receptors or other closely related GPCRs with identifiedendogenous ligands. Schild analyses and radioligand competition assaysrevealed DRD2 affinities that were consistent with those identified forONC201 anticancer activity. In accordance with superior selectivity,ONC201 exhibited a wider therapeutic window compared to otherantipsychotics. In support of the hypothesis that selectively targetingD2-like receptors yields superior anti-cancer efficacy, combinedDRD2/DRD1 inhibition was found to be inferior to DRD2 inhibition alone.ONC201 exhibited a very slow association rate for DRD2 relative toantipsychotics, whereas the dissociation rate was similar to atypicalantipsychotics that are better tolerated clinically. Shotgun mutagenesisacross 350 amino acids of DRD2 identified 8 residues critical forONC201-mediated antagonism of DRD2-induced calcium flux. Severalresidues were not conserved among other dopamine receptors, suggesting apotential role in conferring ONC201 selectivity. Consistent withcompetitive inhibition, several mutated residues were within theorthosteric binding site (OBS), however, two distal residues wereidentified outside of the OBS suggesting a secondary binding pocket. Insummary, receptor pharmacology of ONC201, the first selective DRD2/3antagonist in clinical neuro-oncology, may explain its uniqueselectivity, safety, and anti-cancer activity in clinical trials.

Example 21: Imipridone Family Member ONC206 Suppresses Glioma Stem CellMaintenance

Imipridones selectively target G protein-coupled receptors (GPCRs) thatcontrol critical signaling pathways in various cancer cells. AberrantGPCR overexpression has been implicated in tumorigenesis. ONC201, afirst generation imipridone that directly antagonizes dopamine receptorD2 (DRD2), continues to be evaluated in clinical trials for advancedcancers.

METHODS & RESULTS: Here, ONC206, an ONC201 analog that shares the sameimipridone core chemical structure and selective DRD2 antagonism,potently inhibits patient-derived glioma stem cell (GSC) populations. Insilico analysis of a glioma patient database led to investigation ofDRD2 signaling in glioma; alteration of DRD2 mRNA expression wasdirectly connected to global gene expression change in the gliomapatient database. CellTiter-Glo cell viability assay showed thatexposure to ONC206 in a dose dependent manner preferentially eliminatedGSCs, compared to differentiated glioma cells. Protein array of stemcell markers revealed ONC206 treatment down-regulated protein expressionof oncogenic stem cell markers in the GSCs. Further, in vitro limitingdilution assay and sphere formation analysis showed that ONC206prevented tumor sphere formation and tumor growth. These observationsindicate that ONC206 exhibits promising anti-glioma activity and warrantelucidating the downstream effects of antagonizing DRD2 signaling withONC206 in gliomas.

One skilled in the art will appreciate that changes could be made to theexemplary embodiments shown and described above without departing fromthe broad inventive concept thereof. It is understood, therefore, thatthis invention is not limited to the exemplary embodiments shown anddescribed, but it is intended to cover modifications within the spiritand scope of this invention as defined by the claims. For example,specific features of the exemplary embodiments may or may not be part ofthe claimed invention and features of the disclosed embodiments may becombined. Unless specifically set forth here, the terms “a”, “an” and“the” are not limited to one element but instead should be read to mean“at least one.”

It is to be understood that the figures and descriptions may have beensimplified to focus on elements that are relevant for a clearunderstanding, while eliminating, for purposes of clarity, otherelements that one skilled in the art will appreciate may also comprise aportion of the invention. However, because such elements are well knownin the art, and because they do not necessarily facilitate a betterunderstanding of the invention, a description of such elements is notprovided herein.

Further, to the extent that a method does not rely on the particularorder of steps set forth, the particular order should not be construedas a limitation on the claims. Claims directed to a method should not belimited to performance of the steps in the order written, and oneskilled in the art can readily appreciate that they can be varied andstill remain within the spirit and scope of this invention.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entirety here.

What is claimed is:
 1. A method of treating cancer in a subject in needthereof, comprising: administering to the subject in need of suchtreatment a pharmaceutical composition comprising a therapeuticallyeffective amount compound (1)

or a pharmaceutically acceptable salt thereof, wherein the cancer is aglioma having a histone H3 K27M mutation.
 2. The method according toclaim 1, wherein the glioma is selected from the group consisting of adiffuse intrinsic pontine glioma, a diffuse midline glioma, a spinalcord glioma, a thalamic glioma, a brainstem glioma, and a cerebellarglioma.
 3. The method according to claim 1, wherein the glioma is not aspinal cord tumor.
 4. The method according to claim 1, wherein thehistone H3 K27M mutation in the glioma is H3.3 K27M or H3.1 K27M.
 5. Themethod according to claim 1, wherein the histone H3 K27M mutation in theglioma is in one or more histone genes selected from H3F3A, H3F3B,HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G,HIST1H3H, HIST1H3I, or HIST1H3J.
 6. The method according to claim 1,wherein in cancerous tissue DRD2 is overexpressed, DRD5 isunderexpressed, or both.
 7. The method according to claim 1, wherein thesubject is a human.
 8. The method according to claim 1, wherein thesubject is a domesticated pet.
 9. The method according to claim 1,wherein the subject is a pediatric subject.
 10. A method of treatingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof,wherein the cancer has a histone H3 mutation.
 11. The method accordingto claim 10, wherein the cancer is selected from the group consisting ofa central nervous system tumor, a brain tumor, a peripheral nervoussystem tumor, a pheochromocytoma, a paraganglioma, an adrenal corticalcarcinoma, an adrenal tumor, and a neuroendocrine tumor.
 12. The methodaccording to claim 10, wherein the cancer is selected from the groupconsisting of meningioma, ependymoma, glioma, neuroblastoma, and diffuseintrinsic pontine glioma.
 13. The method according to claim 10, whereinthe cancer is selected from the group consisting of a diffuse midlineglioma, a spinal cord glioma, a thalamic glioma, a brainstem glioma, anda cerebellar glioma.
 14. The method according to claim 10, wherein thehistone H3 mutation is H3.3 K27M or H3.1 K27M.
 15. The method accordingto claim 10, wherein the cancer has a K27M mutation in one or morehistone genes selected from H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C,HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J.16. The method according to claim 10, wherein DRD2 is overexpressed incancerous tissue.
 17. The method according to claim 10, wherein thesubject is a human.
 18. The method according to claim 10, wherein thesubject is a domesticated pet.
 19. The method according to claim 10,wherein the compound is ONC201.
 20. The method according to claim 10,wherein the subject is a pediatric subject.
 21. A method of treatingcancer in a subject in need thereof, comprising: administering to thesubject in need of such treatment a pharmaceutical compositioncomprising a therapeutically effective amount a compound of formula (10)or an analog thereof, or a pharmaceutically acceptable salt thereof,wherein the cancer is a midline glioma.
 22. The method according toclaim 21, wherein the cancer is selected from the group consisting of adiffuse intrinsic pontine glioma, a diffuse midline glioma, a spinalcord glioma, a thalamic glioma, a brainstem glioma, and a cerebellarglioma.
 23. The method according to claim 21, wherein the cancer is nota spinal cord tumor.
 24. The method according to claim 21, wherein thecancer has a histone H3 mutation, wherein the histone H3 mutation isH3.3 K27M or H3.1 K27M.
 25. The method according to claim 21, whereinthe cancer has a histone H3 K27M mutation in one or more histone genesselected from H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D,HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J.
 26. Themethod according to claim 21, wherein DRD2 is overexpressed in canceroustissue.
 27. The method according to claim 21, wherein the subject is ahuman.
 28. The method according to claim 21, wherein the subject is adomesticated pet.
 29. The method according to claim 21, wherein thecompound is ONC201.
 30. The method according to claim 21, wherein thesubject is a pediatric subject.